JP2009180697A - Analysis plate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis plate having a quantitative chamber for accurately quantitatively determining a biological sample. <P>SOLUTION: This analysis plate is formed of the quantitative chamber that is disposed so as to come into contact with the liquid feed channel for feeding the biological sample and quantitatively determining the biological sample. The quantitative chamber has a partition for separating the liquid feed channel from the quantitative chamber in the center section, and connection channels for connecting the liquid feed channel to the quantitative chamber at both ends of the partition. The width of the connection channels is narrower than that of the liquid feed channel coming into contact with the partition. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an analysis plate for discriminating a biological sample by moving a DNA, protein or other biological sample in a buffer and detecting its transport reaction.

  A common biological sample is DNA, and for this DNA, currently the same SNPs (short for single nucleotide polymorphism, generally translated as “single nucleotide polymorphism”), one code (one base) in a gene. It is a collective term for differences.) Since there is absolutely no human being, it has attracted attention because it can completely identify an individual. Currently, as a method for examining SNPs, sequencing (determination of base sequence) in which the base sequence of DNA is directly read from the end is most commonly used. There are several reports on the sequencing method, but the most common method is dideoxy sequencing (Sanger method). Sequencing is based on a technique that separates and identifies the difference in length of one base length by denaturing polyacrylamide gel electrophoresis with high resolution or capillary electrophoresis in any method including the Sanger method. This is true.

  As shown in FIG. 7A, the flow path pattern is formed on concentric circles with the center of gravity 30 of the analysis plate 28 as the center of the flow path for SNPs using this capillary electrophoresis. The outer shape of the analysis plate 28 is an 8 cm square, three of the four corners are provided with R, and the remaining one is chamfered. Next, as shown in FIG. 7B, the sample flow path includes the sample injection section 5, the flow path 14, the chamber 13, the flow path 16, the liquid supply flow path 17A, the liquid supply flow path 17B, the flow path 18, The flow path 19, the sample holding section 15, the buffer section 21, the quantitative chamber 22, the first connection flow path 24 </ b> A, and the second connection flow path 24 </ b> B are configured.

  Next, as a method of measuring SNPs, the analysis plate 28 is fixed to a motor or the like, and rotated at 2000 rpm around the center of gravity 30 (first rotation). The DNA conjugate 32 in the DNA conjugate injection part filled with 3 μl with a pipetter or the like is filled to the positive electrode part 6 and the negative electrode part 7 up to about 70% respectively, fills the flow path 8 and moves to the quantitative chamber 22. The liquid level height of the DNA conjugate 32 existing in the positive electrode part 6 and the negative electrode part 7 and the liquid level height of the quantitative chamber 22 are on the same circle with the center of gravity 30 as the center. Similarly, the DNA sample 27 in the DNA sample injection section 5 filled with 1 μl passes through the flow path 14, passes through the chamber section 13, the flow path 16, and the flow path 18, and is a sample holding section without an air vent hole. Reach up to 15.

  It will be in the state filled with the DNA sample 27 with which the sample holding | maintenance part 15 was filled, and the pressurized air. The pressurized air in the sample holder 15 maintains a state where such centrifugal force and applied pressure are in equilibrium only during rotation. Next, the rotation is suddenly stopped while the movement of the DNA conjugate 32 and the DNA sample 27 is stopped. Then, the DNA sample 27 in the sample holder 15 passes through the flow path 18, the liquid supply flow path 17 A, and the first connection flow path 24 A due to the pressure of air and reaches the quantitative chamber 22, the liquid supply flow path 17 B, and the buffer section 21 (sample) Filling process).

  In particular, the quantitative chamber 22 comes into contact with the DNA conjugate 32 filled with an amount necessary for the examination. Next, the analysis plate 28 is rotated again for a few seconds at a medium speed (second rotation), and a specific amount is quantified in the quantification chamber 22. At this time, it is important to moderate the deceleration. The excess DNA sample 27 filled in the liquid supply channels 17A and 17B and the liquid supply channel 25 is excluded to the chamber unit 13, and the DNA sample 27 is quantified in the quantitative chamber 22. The quantified DNA sample 27 is electrically insulated from other DNA samples 27 (see, for example, Patent Document 1).

Next, FIG. 8 in which the liquid supply channels 17A and 17B and the quantitative chamber 22 are enlarged is shown. In the investigation method described above, when the DNA sample 27 is introduced into the quantitative chamber 22 by air pressure, the DNA sample 27 passes through the liquid supply channel 17A and is introduced into the liquid supply channel 25 and the quantitative chamber 22, and then the liquid supply flow. Fill road 17B. In addition, after the DNA sample 27 is introduced into the quantitative chamber 22, the extra DNA sample 27 in the liquid supply flow paths 17 </ b> A and B other than the quantitative chamber 22 is transferred to the flow path 18 and the flow path 19 by the second rotation. Returned to At this time, a partition wall 23 is provided in order to prevent the DNA sample 27 from flowing out from the quantitative chamber 22 to the liquid feeding channel 17A. As a result, a certain amount of DNA sample 27 can be secured in the quantitative chamber 22 (see, for example, Patent Document 2).
JP 2006-220531 A JP 2006-153562 A

  In the conventional configuration, the DNA sample can be quantified in the quantification chamber by providing the partition wall. However, if the wall surface inside the flow path is in a hydrophilic state, a part of the DNA sample to be held in the quantification chamber. Has been transferred to the chamber portion and cannot be quantified.

  The present invention solves the above-mentioned conventional problems, and an object thereof is to provide an analysis plate having a quantitative chamber capable of accurately quantifying a biological sample.

  In order to solve the above-mentioned conventional problems, the analysis plate of the present invention is an analysis comprising a quantitative chamber for quantifying the biological sample provided so as to be in contact with a liquid supply channel for supplying the biological sample. In the plate, the metering chamber has a partition for separating the liquid feeding channel and the metering chamber at a central portion thereof, and a connection for connecting the liquid feeding channel and the metering chamber to both ends of the partition. And the width of the connection flow path is narrower than the width of the liquid supply flow path in contact with the partition wall.

  According to the analysis plate of the present invention, a fixed amount of DNA sample is held in the quantitative chamber by providing the width of the liquid supply channel on the opposite side of the quantitative chamber across the partition wall as compared with the second connection channel. Only the extra DNA sample can be excluded.

  Hereinafter, embodiments of the analysis plate of the present invention will be described in detail with reference to the drawings.

(Embodiment 1)
The analysis plate 28 according to Embodiment 1 of the present invention will be described with reference to FIGS.

  FIG. 1A shows the analysis plate 28 according to the first embodiment. The analysis plate 28 has grooves formed on the surface thereof, and is used as the analysis plate 28 by sticking the film 26 on this surface. The groove is a flow path or a chamber constituted by the flow path pattern 1, and the DNA sample 27 introduced into the flow path is transferred to the previous flow path by the centrifugal force when the analysis plate 28 is rotated.

  FIG. 1B is an enlarged view of the flow path pattern 1 formed on the analysis plate 28. In FIG. 1B, the buffer injection port 2 is a buffer injection port 2 for injecting a DNA conjugate 32 as a buffer, and the buffer injection unit 3 is a buffer injection unit for temporarily holding the injected buffer. 3. The sample injection port 4 is a sample injection port 4 for injecting a DNA sample 27, and the sample injection unit 5 is a sample injection unit 5 for temporarily holding the injected DNA sample 27. The positive electrode insertion portion 6 and the negative electrode insertion portion 7 are connected via a flow path 8, and are further connected to the buffer injection part 3 by flow paths 9 and 10, respectively. The chamber part 13 is connected to the sample injection part 5 by a flow path 14. Reference numeral 15 denotes a sample holding unit which is connected to the chamber unit 13 by a flow channel 16 and a second flow channel 18. Further, the liquid flow path 17 </ b> A and the flow path 19 are also connected to the flow path 18.

  The buffer unit 21, the liquid supply channels 17A and 17B, the channel 18 and the channel 19 are connected to the quantitative chamber 22, the first connection channel 24A, the second connection channel 24B and the liquid supply channel 25, respectively. Further, the buffer section 21 can be vented by the flow path 20 connecting the sample injection section 5 and the buffer section 21. The first connection channel 24A and the second connection channel 24B are formed by providing the partition wall 23 at a portion where the fixed amount chamber 22 and the liquid supply channel 25 are connected. The buffer injection part 3, the flow path 9, the flow path 10, the positive electrode part 6, the negative electrode part 7 and the flow path 8 are closed flow paths, and the buffer flows through the closed flow paths, It is configured as a flow path for the buffer.

  Further, the sample injection unit 5, the channel 14, the chamber unit 13, the channel 16, the liquid supply channels 17A and 17B, the channel 18, the channel 19, the sample holding unit 15, the channel 20, the buffer unit 21, and the quantitative chamber 22, the partition wall 23, the first connection channel 24A, the second connection channel 24B, and the liquid feed channel 25 are also closed channels, and are configured as channels for DNA samples. The flow path through which the DNA sample 27 passes and the flow path through which the DNA conjugate 32 passes merge in the quantitative chamber 22, and the DNA sample 27 quantified in the quantitative chamber 22 is used for measurement.

  The wall surface constituting the flow path pattern 1 is subjected to a hydrophobic treatment. Hydrophobic treatment prevents sample liquid from being introduced by capillary force from the chamber to the flow channel, and from the flow channel to the chamber, and can be advanced through the flow channel using external force such as centrifugal force, which hinders inspection. It can be operated without any problems. Hydrophobic treatment includes a method of treating biodegradable hydrophobic polyester with high-temperature and high-pressure water.

  FIG. 2 shows an enlarged view of the liquid feeding flow path 25 including the metering chamber 22 in Embodiment 1 of the present invention. This is an enlarged view of the fixed quantity chamber 22 and the liquid supply flow path 25, the liquid supply flow paths 17A and 17B, the partition wall 23, the first connection flow path 24A, and the second connection flow path 24B connected to the fixed measurement chamber 22 in FIG. It is a thing. The liquid supply channels 17A and B carry the DNA sample 27 from the liquid supply channels 17A to 17B during the sample filling process, and when the amount is quantified in the quantitative holding unit 22, from the liquid supply channels 17B to 17A. Have a role. The partition wall 23 is provided for quantifying the DNA sample 27 held in the quantification chamber 22. A liquid feed channel 25 having a width wider than the width of the second connection channel 24B is provided opposite to the quantitative chamber 22 with the partition wall 23 interposed therebetween. This is to prevent the DNA sample 27 to be quantified in the quantification chamber 22 from being excluded by the second rotation, and thus the liquid supply channels 17A and 17B and the liquid supply channel 25 after the second rotation. Only the extra DNA sample 27 can be excluded.

  Details of the analysis plate 28 used in this example are shown in FIG. FIGS. 3A and 3B show a cross section AA ′ in the vicinity of the liquid supply channels 17A and 17B and the quantitative chamber 22 in the analysis plate 28 according to Embodiment 1 of the present invention. In FIG. 3B, the right side is a flow path forming surface. The hatched portion is the analysis plate 28. As shown in FIG. 3A, the widths of the liquid feeding channels 17A and 17B and the liquid feeding channel 25 are 0.1 mm, and the width of the quantitative chamber is 0.3 mm. The width of the first connection channel 24A and the second connection channel 24B is set to 0.15 mm, and the width of the liquid supply channel 25 is set to be wider than that of the second connection channel 24B. As shown in FIG. 3B, the depths of these flow paths are both 0.08 mm. The width of the liquid feeding flow path 25 of the present embodiment is set to 1 mm or less because the sample injection port 4 and the sample injection part 5 are arranged on the rotation center side. However, the width of the liquid flow path 25 can be changed as appropriate depending on the amount of the DNA sample 27 to be introduced.

  FIG. 4 shows the force acting in the vicinity of the quantitative chamber during the second rotation. FIG. 4A shows the state of the force observed during the second rotation when the surface of the analysis plate 28 is in the hydrophobic state, and FIG. 4B shows the state of the second rotation when the surface of the analysis plate 28 is in the hydrophilic state. The state of the force seen in is shown. If the analysis plate 28 is in a hydrophobic state, the liquid also has a large force to become spherical due to van der Waals force. Therefore, even if the widths of the liquid supply flow channels 17A and B are narrower than the widths of the first connection flow channel 24A and the second connection flow channel 24B, the force that pushes out to the outer periphery by centrifugal force and the force that flows in the circumferential direction by inertia force By working, it is divided into a DNA sample 27 held in the quantitative chamber 22 and a DNA sample 27 passing through the liquid feeding flow path 17A. However, if the analysis plate 28 is in a hydrophilic state, the liquid flows in such a way that the capillary force acts on the wall.

  The capillary force acts to cause the DNA sample 27 to flow in the narrow flow path, but the DNA sample 27 flows from the liquid feed flow path 17B to 17A by the inertial force due to rotation, and flows in the direction of the quantitative chamber 22 by the centrifugal force. And At this time, the fluid flows preferentially in the first connection channel 24A and the second connection channel 24B having a wider channel width than the liquid feeding channels 17A and B having a narrow channel width. Therefore, even if the partition wall 23 is provided, the DNA sample 27 in the quantitative chamber 22 is excluded so as to be pushed out from the liquid feeding channel 17B. Therefore, it is necessary to secure a wider flow path than the first connection flow path 24A and the second connection flow path 24B in the first connection flow path 24A and the second connection flow path 24B. On the opposite side of the quantitative chamber 22, a liquid feeding passage 25 having a width wider than that of the second connection passage 24 </ b> B was provided.

  In FIG. 5, the effect by having provided the liquid feeding flow path 25 which has the wide width in the analysis plate 28 of this invention is shown. FIG. 5A shows a case where the liquid connection flow path 25 is wider than the width of the second connection flow path 24B. FIG. 5B shows a liquid supply flow path 25 which is narrower than the width of the second connection flow path 24B. This is the case with In both cases, the surface of the analysis plate 28 is in a hydrophilic state. 5 (a) and 5 (b) show the common flow changes (1) to (3). (1) shows a state in which the DNA sample 27 is filled in the quantitative chamber 22, (2) shows a state in which the DNA sample 27 is quantified by the second rotation, and (3) shows a state in which the second rotation is stopped. Yes.

  FIG. 5B (3) shows a state in which the DNA sample 27 in the quantitative chamber 22 is removed. This occurs because the extra DNA sample 27 has passed through the quantitative chamber 22 without passing through the liquid feeding channel 25 on the partition wall 23. That is, since the surface of the analysis plate 28 is in a hydrophilic state and the width of the liquid feeding flow path 25 above the partition wall 23 is narrower than the width of the connection flow path 24B, the width easily flows due to the centrifugal force and inertial force due to rotation. This is because the second connection channel 24B having a large width is passed.

  Next, (3) in FIG. 5A shows a state in which the DNA sample 27 in the quantitative chamber 22 is quantified without being removed. This occurs because excess DNA sample 27 staying in the liquid supply channels 17A and 17B and the liquid supply channel 25 has passed through the liquid supply channel 25. Therefore, if the width of the liquid supply channel 25 is wider than the width of the connection channel 24B, it indicates that the DNA sample 27 can be quantified in the quantification chamber 22 even when the surface of the analysis plate 28 is in a hydrophilic state.

  FIG. 6 shows a similar shape of the liquid feeding passage 25. FIG. 6 shows a case where the shape of the liquid supply passage 25 is a semicircular arc. Even in the shape as shown in the figure, by providing a wider width of the liquid supply flow path 25 than the first connection flow path 24A and the second connection flow path 24B, the DNA sample 27 in the vicinity of the quantitative chamber 22 is eliminated, It was possible to hold the DNA sample 27 only to confirm the effect.

  As described above, according to the first embodiment, after the second rotation, only the extra DNA sample 27 remaining in the liquid feeding channels 17A and 17B is removed by centrifugal force, and a specific amount of DNA is placed in the quantification chamber 22. The sample 27 can be accurately quantified.

  The analysis plate of the present invention is useful as a gene analyzer for discriminating biological samples such as DNA samples.

Flow path pattern diagram of analysis plate in Embodiment 1 of the present invention The perspective view of the liquid feeding flow path part containing the fixed_quantity | quantitative_assay chamber in Embodiment 1 of this invention. The figure which shows the flow path pattern in the presence or absence of the liquid feeding flow path of the analysis plate in Embodiment 1 of this invention. The figure shown about the force applied to the liquid feeding flow path vicinity at the time of the 2nd rotation in Embodiment 1 of this invention Sectional drawing of the vicinity of the flow path and chamber in the analysis plate of Embodiment 1 of this invention The figure which shows the similar shape of the liquid feeding flow path in Embodiment 1 of this invention Diagram showing the configuration of a conventional analysis plate Diagram showing the configuration of a conventional analysis plate

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Channel pattern 2 Buffering agent inlet 3 Buffering agent injection part 4 Sample injection port 5 Sample injection part 6 Positive electrode part 7 Negative electrode part 8 Channel 9 Channel 10 Channel 11 Channel 12 Channel 12 Channel 13 Chamber part 14 Flow Path 15 Sample holding section 16 Flow path 17A Liquid feed flow path 17B Liquid feed flow path 18 Flow path 19 Flow path 20 Flow path 21 Buffer section 22 Metering chamber 23 Bulkhead 24A First connection flow path 24B Second connection flow path 25 Flow path 26 Film 27 DNA sample 28 Analysis plate 29 Hole for fixing rotating part 30 Plate center of gravity 31 Positioning hole 32 DNA conjugate

Claims (7)

In an analysis plate comprising a quantitative chamber for quantifying the biological sample provided so as to be in contact with a liquid supply flow path for supplying a biological sample,
The metering chamber has a partition for separating the liquid feeding channel and the metering chamber at a central portion thereof, and a connection channel for connecting the liquid feeding channel and the metering chamber to both ends of the partition. Have
An analysis plate in which the width of the connection channel is narrower than the width of the liquid supply channel in contact with the partition wall. The connection channel is a first connection channel that is first opened with respect to the liquid feeding direction of the biological sample that is first fed to the solution feeding channel, and one of the connection channels is a second connection channel. The analysis plate according to claim 1. The analysis plate according to claim 2, wherein a width of the second connection channel is narrower than a width of the liquid supply channel in contact with the partition wall. The analysis plate according to claim 1, wherein the liquid flow path and the inner wall of the quantitative chamber are subjected to a hydrophobic treatment. The analysis plate according to claim 1, wherein the analysis plate is provided with a rotation shaft so as to be rotatable. The analysis plate according to claim 5, wherein the rotation shaft is provided in a direction from the partition wall of the quantitative chamber to the liquid supply flow path. The analysis plate according to claim 5, wherein the rotation shaft is provided at a position where the biological sample is supplied to the liquid supply channel by rotating the analysis plate.

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