JP4944603B2 - Biochemical reaction vessel - Google Patents

Description

  The present invention provides a probe carrier such as a microarray that can be suitably used when a test is performed on the presence or absence of a gene or protein derived from a pathogenic bacterium in a specimen such as blood and used as a material for determining the health condition of a test subject. The present invention relates to a biochemical reaction vessel provided. More specifically, the present invention relates to the structure of a biochemical reaction vessel for at least uniforming the flow rate of the liquid flowing in the reaction chamber.

  Many methods using a hybridization reaction using a probe carrier typified by a DNA microarray have been proposed as a method for quickly and accurately analyzing a nucleic acid base sequence and detecting a target nucleic acid in a nucleic acid sample. A DNA microarray is a probe having a base sequence complementary to a target nucleic acid fixed on a solid phase such as a bead or a glass plate at a high density. The detection of a target nucleic acid using this probe is generally as follows. It has a process.

  As a first step, a target nucleic acid is amplified by an amplification method typified by a PCR method. Specifically, first, first and second primers are added to a nucleic acid sample, and a temperature cycle is applied. The first primer specifically binds to a part of the target nucleic acid, and the second primer specifically binds to a part of the nucleic acid complementary to the target nucleic acid. When the double-stranded nucleic acid containing the target nucleic acid is bound to the first and second primers, the double-stranded nucleic acid containing the target nucleic acid is amplified by the extension reaction. After the double-stranded nucleic acid sufficiently containing the target nucleic acid is amplified, a third primer is added to the nucleic acid sample and subjected to a temperature cycle. The third primer is labeled with an enzyme, a fluorescent substance, a luminescent substance, or the like, and specifically binds to a part of the nucleic acid complementary to the target nucleic acid. When a nucleic acid complementary to the target nucleic acid and the third primer are bound, the target nucleic acid labeled with an enzyme, a fluorescent substance, a luminescent substance or the like is amplified by an extension reaction. As a result, a labeled target nucleic acid is produced when the target nucleic acid is contained in the nucleic acid sample, and a labeled target nucleic acid is not produced when the target nucleic acid is not contained in the nucleic acid sample.

  As a second step, the nucleic acid sample is brought into contact with a DNA microarray and subjected to a hybridization reaction with a probe of the DNA microarray. When there is a target nucleic acid complementary to the probe, the probe and the target nucleic acid form a hybrid.

  As a third step, the target nucleic acid is detected. Whether the probe and the target nucleic acid form a hybrid can be detected by a labeling substance of the target nucleic acid, thereby confirming the presence or absence of a specific base sequence.

  DNA microarrays utilizing this hybridization reaction are expected to be applied to medical diagnosis for identifying pathogenic bacteria and genetic diagnosis for examining patient constitutions. However, the nucleic acid amplification, hybridization, and detection steps are often performed by individual apparatuses, and the work is complicated, requiring a considerable amount of time for diagnosis. In particular, in a configuration in which a hybridization reaction is performed on a slide glass, the probe fixing surface is exposed, and there is a possibility that the probe may be lost or contaminated when a finger touches the slide glass. Must be done carefully. In order to solve these problems, several biochemical reaction vessel structures have been proposed in which a DNA microarray is provided in a reaction chamber, a hybridization reaction is performed in the reaction chamber, and detection is possible thereafter.

  Japanese National Publication No. 10-505410 discloses a structure and a manufacturing method for forming a cavity. Japanese Patent Application Laid-Open Nos. 2003-302399 and 2004-093558 disclose a chamber structure for preventing bubbles from remaining during the initial filling of the liquid. Furthermore, Japanese Patent Application Laid-Open No. 2002-243748 discloses a structure for forming a uniform spread and flow of a liquid.

In such a biochemical reaction vessel structure, the volume of the reaction chamber is as small as about several tens of μL, the height of the reaction chamber is low, and the reaction chamber has a planar space. As a result, the amount of reagents used is small, and a laminar flow is generated in the reaction chamber. Furthermore, in order to increase the efficiency of the hybridization reaction between the probe on the solid phase and the target nucleic acid, the liquid is preferably stirred in the reaction chamber. As the simplest method, there is a method in which the liquid in the reaction chamber is swung by pushing and pulling the liquid at the inlet.
Japanese National Patent Publication No. 10-505410 JP 2003-302399 A JP 2004-093558 JP JP 2002-243748 A

  An example of a biochemical reaction vessel is shown in FIG. It is assumed that the reaction chamber 103 of the biochemical reaction vessel 110 is filled with a liquid. When the liquid is further fed from the inlet 106, the flow velocity 122 near the center becomes faster when the flow velocity 122 near the center of the reaction chamber 103 is compared with the flow velocity 121 and 123 near the ends. Therefore, when the liquid in the reaction chamber 103 is swung by pushing and pulling the liquid at the inlet 106 or the outlet 107, the frequency of contact between the probe on the solid phase and the target nucleic acid is near the center and near the end of the reaction chamber 103. Then it will be different. In addition, after the hybridization reaction is completed, a washing solution is flowed into the reaction chamber 103 in order to remove unreacted nucleic acids. Also at this time, the flow rate 122 near the center of the reaction chamber 103 and the flow rates 121 and 123 near the ends are different, so the rate at which unreacted nucleic acid is removed and the target nucleic acid that has reacted with the probe on the solid phase is stripped. Probabilities are different. As a result, the brightness at the time of detection varies depending on the position of the probe, which may adversely affect the diagnosis result.

  In the configuration disclosed in Japanese Patent Publication No. 10-505410, although a laminar flow is generated in the cavity, there are cases where the difference in flow velocity between the center and the end of the cavity cannot be resolved. In addition, in the configurations of Japanese Patent Application Laid-Open Nos. 2003-302399 and 2004-093558, the liquid spreads uniformly in the chamber at the initial filling of the liquid, but the flow rate in the chamber is increased when the liquid is filled in the chamber. There may be no effect of homogenizing. Further, the configuration of Japanese Patent Application Laid-Open No. 2002-243748 has a complicated structure, and there are cases where there is a limit in reducing the cost for manufacturing the container.

  An object of the present invention is to provide a biochemical reaction vessel having a configuration capable of uniformizing the flow of liquid in a reaction chamber in a planar direction and a vertical direction by adding a simple configuration.

The biochemical reaction container of the present invention includes a reaction chamber for reacting a sample with a probe fixing region for detecting a target nucleic acid, and a first hole provided so as to communicate with one end of both ends of the reaction chamber. And a second hole provided at the other end of the reaction chamber, and the reaction chamber between the first hole and the second hole forms a flow path, and the flow path A biochemical reaction container comprising a fluid resistance portion comprising a probe fixing region and a projecting member for reducing a cross-sectional area of the flow path;
The protruding member includes a region having a tapered shape,
There are the protruding members both between the first hole and the probe fixing region, and between the second hole and the probe fixing region ,
Both the protruding member between the first hole and the probe fixing region and the protruding member between the second hole and the probe fixing region have at least the region having the tapered shape as the probe fixing region. On the side,
The reaction chamber has a ceiling portion and a bottom portion, and the protruding member protrudes in a vertical direction from the ceiling portion to the bottom portion of the reaction chamber, and the taper shape has an interval between the ceiling portion and the bottom portion. The taper shape has an inclination that gradually spreads to the ceiling,
The biochemical reaction vessel , wherein the probe fixing region is provided on a part of the bottom surface .

  According to the present invention, the member for reducing the cross-sectional area of the flow path in the flow path configured to have the first hole that can be used as the inlet, the reaction chamber, and the second hole that can be used as the outlet. By arranging it, the in-plane flow velocity can be controlled. Furthermore, since the fluid resistance portion has a tapered shape, air and liquid fluid after passing through the fluid resistance portion are stabilized, turbulent flow is reduced, and there is an effect that liquid accumulation and air accumulation can be improved.

  Embodiments according to the present invention will be described below with reference to the drawings.

(Embodiment 1)
In this embodiment, in the biochemical reaction vessel characterized in that the reaction chamber has a fluid resistance portion, and the fluid resistance portion includes a region having a tapered shape, the protrusion shape of the fluid resistance portion is optimized, and the liquid An example of controlling the flow of the liquid and improving the liquid pool will be described.

  FIG. 1 is a perspective view showing the structure of a biochemical reaction vessel (hereinafter referred to as a biochemical reaction cassette) according to the first embodiment of the present invention. 2A, 2B, and 2C are a plan view, a cross-sectional view, and an enlarged cross-sectional view showing the structure of the biochemical reaction cassette according to the first embodiment of the present invention. 3 and 4 are plan and vertical sectional schematic views showing the state of the liquid flowing inside the biochemical reaction cassette according to the first embodiment of the present invention.

  The structure of the cassette shown in FIG. 1 or FIG. 2 (a) will be described. The cassette 10 has a configuration in which a glass substrate 11 and a housing 12 made of polycarbonate are joined. In addition, the joining form of the housing | casing part with respect to a glass substrate is not limited to the illustrated example, A various form can be taken. The material of the housing 12 is not limited to polycarbonate, but may be plastic other than polycarbonate, glass, rubber, silicon, or the like. On the joint surface between the glass substrate 11 and the housing 12, a recess having a predetermined cross-sectional shape is provided in the housing 12, and the first buffer chamber 1 and the first slot portion 2 are provided between the glass substrate 11 and the housing 12. The reaction chamber 3, the second slot portion 4, and the second buffer chamber 5 are formed. These parts are arranged adjacent to each other and form a flow path. The bottom surface of each space constituting these portions formed between the glass substrate 11 and the housing 12 is formed of a part of the surface of the glass substrate 11. Here, since the casing 12 is provided with spaces for a buffer chamber, a slot portion, and a reaction chamber, the bottom surfaces of the spaces are the same plane. However, a part or all of the buffer chamber, the slot portion, and the reaction chamber are made of glass. The structure which is provided in the board | substrate 11 and the bottom face of each space does not comprise the same plane may be sufficient.

  As shown in FIG. 2B, the ceiling of each slot is lower than the ceiling of the buffer chamber and the reaction chamber, and has a shape as shown in the figure. The upper part of each slot is the buffer chamber and the reaction. A partition of the chamber is formed.

  As shown in FIG. 2A, a probe fixing region 13 is provided on a part of the surface of the glass substrate 11 on the bottom surface of the reaction chamber 3, and the target nucleic acid is contained in the liquid filled in the reaction chamber 3. Is contained, the target nucleic acid reacts with the probe in the probe fixing region 13. The combination of the target nucleic acid and the probe can be selected according to the detection purpose such as when both of them are DNA.

  The reaction chamber 3 is provided with two external communication holes. In this embodiment, one of them is used as the inlet 6 and the other is used as the outlet 7.

  The liquid was injected into the first buffer chamber 1 from the injection port 6, passed through the first slot portion 2, the reaction chamber 3, the second slot portion 4 and the second buffer chamber 5 in this order, and connected to the second buffer chamber 5. It is discharged out of the cassette 10 from the discharge port 7. That is, a liquid flow path is formed from these portions.

  The dimensions of each chamber used in this embodiment are displayed as coordinates X (width) × Y (length) × Z (height: distance from each bottom surface to the ceiling) in FIG. As shown in FIG. 2A, the value of the width (coordinate X) is constant regardless of the buffer chamber and is 10 mm. The length (coordinate Y) is as follows. 2B and 2C, the first buffer chamber 1 and the second buffer chamber 5 have the same length of 2.0 mm and the reaction chamber 3 has a length of 10 mm. The length of the region where the heights of the first slot portion 2 and the second slot portion 4 are constant is 1.0 mm, and the length of the region where the height is changed is 1.5 mm. The height (coordinate Z) is constant except for the slot portion and is 0.5 mm. The region where the height of the slot portion is constant is 0.1 mm. Moreover, the taper part ((theta) of FIG. 9) of the protrusion member in this embodiment is about 72 degrees.

  Note that the inclination of the tapered portion may be set to such an extent that the intended effect of the present invention is obtained, and can be selected from a range of preferably 30 to 85 °, more preferably 45 to 80 °.

  Next, a method for reacting a target nucleic acid using a cassette will be described. First, a nucleic acid sample is prepared, and the target nucleic acid is amplified according to the method described above as necessary. When the target nucleic acid is present in the nucleic acid sample, a target nucleic acid labeled with a fluorescent substance is generated in the amplification step. Here, the labeling substance is a fluorescent substance, but it may be a luminescent substance or an enzyme. The nucleic acid sample solution is injected into the cassette 10 from the injection port 6 using liquid injection means (not shown). The solution is filled into the first buffer chamber 1, the first slot portion 2, the reaction chamber 3, the second slot portion 4 and the second buffer chamber 5. Almost simultaneously with this, the cassette, the substrate, and the solution are heated, and the hybridization reaction between the target nucleic acid in the solution and the probe on the probe fixing region 13 proceeds. At this time, in order to increase the frequency with which the target nucleic acid in the solution contacts the probe on the probe fixing region 13, the solution is reciprocated in the reaction chamber 3 and stirred. At this time, the first buffer chamber 1, the first slot portion 2, the reaction chamber 3, the second slot portion 4 and the second buffer chamber 5 are always filled with the solution.

  When a solution of a nucleic acid sample is fed from the inlet 6 side for stirring, a flow as shown in FIG. If there is no resistance in the passage of the liquid, the solution flows in a substantially straight line from the inlet 6 toward the outlet 7 as shown in FIG. In the system having the first slot portion 2 as in the present embodiment, as shown in FIG. 3, since it becomes a resistance of the liquid, the solution flows 21, 22, and 23 that spread throughout the first buffer chamber 1 are generated. Arise. As a result, the pressure of the entire first buffer chamber 1 rises, and an equal pressure is applied to the first slot portion 2. Therefore, the solution pushed out from the first slot portion 2 has a uniform flow rate 24, 25 and 26 in the reaction chamber 3. When the solution of the nucleic acid sample necessary for stirring is fed from the inlet 6, the solution of the nucleic acid sample is then fed from the outlet 7 side. Based on the same principle as when the solution is fed from the inlet 6 side, uniform flow velocities 34, 35, and 36 are generated in the reaction chamber 3 as shown in FIG. After the amount of nucleic acid sample solution necessary for stirring is fed from the discharge port 7, the solution of the nucleic acid sample is again fed from the injection port 6. After this, the liquid supply from the discharge port 7 and the liquid supply from the injection port 6 are repeated to stir the solution in the reaction chamber 3. Since a uniform flow rate is generated in the reaction chamber 3, the probe present at any position in the probe fixing region 13 has the same frequency of contact with the target nucleic acid in the nucleic acid sample, and the hybridization reaction depends on the position of the probe fixing region 13. No difference in the degree of progress. The combination of the target nucleic acid and the probe can be selected according to the detection purpose such as when both of them are DNA. The probe fixing region can be formed using a probe carrier such as a DNA chip in which a large number of probes are arrayed and fixed to the carrier.

  When a nucleic acid sample solution is fed from the inlet 6 side, a flow as shown in FIG. 4A occurs in the vertical direction. The solution of the nucleic acid sample injected from the inlet 6 passes through the first slot 2 and then passes as a flow 14. If the slot has a rectangular shape as shown in FIG. 4B, the solution flows as shown by flow 15, turbulence occurs after passing through the slot, and the nucleic acid sample cannot be supplied efficiently, so that the hybridization reaction efficiency Decreases. Due to the slot shape as in this embodiment, the flow of the solution after passing through the slot is also stabilized.

  As described above, in this embodiment, the fluid resistance portion is provided in the reaction chamber, and the shape of the fluid resistance portion is tapered to optimize the protrusion shape of the fluid resistance portion and control the flow of the liquid. In addition, the liquid pool can be improved and the hybridization reaction can be performed efficiently with a small number of samples.

  In this embodiment, since the hybridization reaction was promoted by reciprocating the solution of the nucleic acid sample, two slots were provided on the injection side (slot 2) and the discharge side (slot 4). However, when the reciprocation is not performed, the slot may be either one of the injection side or the discharge side.

  Furthermore, in a system that reciprocates at a high speed to promote the hybridization reaction, the slot shape is configured as shown in FIG. The turbulent flow of the solution as in (b) 15 is eliminated, and the generation of bubbles and the like can be suppressed. Further, considering the liquid pool, even if the slot shape is as shown in FIGS. 5B and 5C, the same effect as the present embodiment can be obtained, and the slot shape is inclined as shown in FIG. May be provided at both ends.

  The probe fixing region may be configured to be detachable from the cassette. This also applies to the following embodiments.

(Embodiment 2)
Hereinafter, a second embodiment of the present invention will be described.

  In this embodiment, a system having a step of discharging the liquid in the reaction chamber at the time of detection (when the chamber is filled with gas) will be described. That is, the present embodiment is an example in which the slot shape is optimized, the airflow is stabilized in the in-plane direction and the vertical direction, and hybridization efficiency is improved and stable luminance is ensured at the time of detection.

  With respect to the cassette used in this embodiment, the description of the same items as those described in the first embodiment will be omitted. 6 (a), 6 (b) and 6 (c) show a biochemical reaction cassette according to the second embodiment of the present invention. A structural difference from the first embodiment is a slot shape.

  The slot shape of this embodiment is a shape like the 1st slot 20 and the 2nd slot 21 which are shown in Drawing 6 (b) and (c). The height of the slot tips of the first slot 20 and the second slot 21 is 0.1 mm. The height of the chamber is 0.5 mm as in the first embodiment. In addition, the inclination (θ in FIG. 9) of the tapered portion of the protruding member in the present embodiment is also about 72 °.

  Next, a method for reacting a target nucleic acid using a cassette will be described. A solution of the nucleic acid sample is injected into the cassette 10 from the injection port 6 using a liquid injection means (not shown). The solution is filled in the first buffer chamber 1, the first slot portion 20, the reaction chamber 3, the second slot portion 21, and the second buffer chamber 5. At the same time, the cassette, the substrate, and the solution are heated, and the hybridization reaction between the target nucleic acid in the solution and the probe on the probe fixing region 13 proceeds. At this time, in order to increase the frequency with which the target nucleic acid in the solution contacts the probe on the probe fixing region 13, the solution is reciprocated in the reaction chamber 3 and stirred. At this time, the first buffer chamber 1, the first slot portion 2, the reaction chamber 3, the second slot portion 4 and the second buffer chamber 5 are always filled with the solution.

  As in Embodiment 1, when a solution of a nucleic acid sample is fed from the inlet 6 side for stirring, a flow as shown in FIG. When a nucleic acid sample solution is fed from the inlet 6 side, a flow as shown in FIG. 4A occurs in the vertical direction after passing through the slot. After the hybridization reaction, the fluorescent substance or the like is removed from the reaction chamber 3 with a cleaning solution, and then the reaction chamber 3 is dried with air. In this case, the air flow is also as shown in FIGS. 3 (a) and 4 (a). By making the slot shape as described above, it was possible to secure stable luminance even when fluorescence was observed by the detector after the reaction chamber 3 was dried.

  As described above, in this embodiment, the slot shape is optimized for a system having a process of discharging the liquid in the reaction chamber at the time of detection (when the inside of the chamber is filled with gas), and the airflow is also improved. By stabilizing the inward and vertical directions, it was possible to improve hybridization efficiency and ensure stable brightness during detection.

  In this embodiment, since the hybridization reaction was promoted by reciprocating the solution of the nucleic acid sample, two slots were provided on the injection side (slot 2) and the discharge side (slot 4). However, when the reciprocation is not performed, the slot may be either one of the injection side or the discharge side.

  Furthermore, in a system that reciprocates at a high speed to promote the hybridization reaction, the slot shape is configured as shown in FIGS. However, the turbulent flow of the solution as shown in FIG. 4 (b) 15 can be eliminated, and the generation of bubbles and the like can be suppressed.

  In the system having air drying, the same effect as that of the present embodiment can be obtained even when the slot shape is as shown in FIGS. 7 (a), (b), (c), and (d).

It is a perspective view explaining the structure of the biochemical reaction cassette which concerns on the 1st Embodiment of this invention. It is the top view, sectional drawing, and expanded sectional view explaining the structure of the biochemical reaction cassette which concerns on the 1st Embodiment of this invention. It is a figure explaining the flow of the liquid of a plane direction in the biochemical reaction cassette which concerns on the 1st Embodiment of this invention. It is a figure explaining the flow of the liquid of the perpendicular direction in the biochemical reaction cassette which concerns on the 1st Embodiment of this invention. FIG. 6 is a view of another slot shape for achieving the first embodiment of the present invention. It is the top view, sectional drawing, and expanded sectional view explaining the structure of the biochemical reaction cassette which concerns on the 2nd Embodiment of this invention. FIG. 6 is another slot shape diagram for achieving the second embodiment of the present invention. They are the perspective view explaining the structure of the biochemical reaction cassette of a prior art example, the top view and sectional drawing explaining a structure. It is typical sectional drawing explaining the inclination angle of the taper-shaped part of a protrusion member.

Explanation of symbols

1 First buffer chamber 2, 16, 18, 30, 20, 22, 24, 26, 28 First slot 3, 103 Reaction chamber 4, 17, 19, 21, 23, 25, 27, 29, 31 Second Slot portion 5 Second buffer chamber 6, 106 Inlet 7, 107 Discharge port 10, 110 Cassette 11, 111 Glass substrate 12, 112 Case 13, 113 Probe fixing region 51 Buffer chamber 52 Slot portion

Claims (4)

A reaction chamber for reacting a sample with a probe fixing region for detecting a target nucleic acid, a first hole provided to communicate with one end of both ends of the reaction chamber, and the other end of the reaction chamber The reaction chamber between the first hole and the second hole forms a flow path, the probe fixing region in the flow path, and the A biochemical reaction vessel comprising a fluid resistance portion comprising a protruding member that reduces the cross-sectional area of the flow path,
The protruding member includes a region having a tapered shape,
There are the protruding members both between the first hole and the probe fixing region, and between the second hole and the probe fixing region ,
Both the protruding member between the first hole and the probe fixing region and the protruding member between the second hole and the probe fixing region have at least the region having the tapered shape as the probe fixing region. On the side,
The reaction chamber has a ceiling portion and a bottom portion, and the protruding member protrudes in a vertical direction from the ceiling portion to the bottom portion of the reaction chamber, and the taper shape has an interval between the ceiling portion and the bottom portion. The taper shape has an inclination that gradually spreads to the ceiling,
The biochemical reaction vessel, wherein the probe fixing region is provided on a part of the bottom surface . 2. The biochemical reaction container according to claim 1, wherein the bottom portion is formed of a plane intersecting with a protruding direction of the protruding member in the vertical direction, and the probe fixing region is provided in a part of the plane. A projecting member between the first hole and the probe fixing region includes a region having the tapered shape on both the first hole side and the probe fixing region side; and
3. The projecting member between the second hole and the probe fixing region includes a region having the tapered shape on both the second hole side and the probe fixing region side. Biochemical reaction vessel. The biochemical reaction container according to any one of claims 1 to 3, wherein an angle of the inclination of the tapered shape with respect to a vertical direction is selected from a range of 45 to 80 °.

Images