1. Field of the Invention
The present invention relates to a simple plate capable of performing a blood test, a urine test, and a DNA test by a medical institution or an individual, and more specifically, to a plate and a test method using the same capable of performing a test on test particles filled inside a flow path with high accuracy.
2. Description of the Related Art
Recently, a testing chip for a collected material from the human body, such as blood or urine, has been increasingly developed. For example, a DNA chip where multiple kinds of DNA fragments (referred to as probes or reagents) are attached on a substrate made of, for example, glass can detect plural kinds of specific genes (for example, cancer gene) from genes (referred to as a test sample or target) collected from the human body at one time.
When the detection of biological molecules, which has been conventionally performed by a test tube, a dropper, an agitator or the like, is performed on the chip, a test can be performed at high speed, and a test process can be simplified, which has attracted attention.
In the meantime, a testing chip is mainly developed as a research chip for a university or a research institution at this time, but it is expected in the future that a simple testing chip for a medical institution or an individual will be commercialized.
The testing chip is composed of a plate substrate having a flow path formed in a groove shape therein and a lid body which is formed on the plate substrate to be joined to the plate substrate.
In a test method, probes having specific base sequences are disposed inside the flow path, and DNA (test sample) collected from the human body is labeled with a fluorescent dye to flow inside the flow path. Then, if the test sample includes DNA having complementary base sequences to the probe, the DNA is hybridized with the probe to be captured.
For example, the probe can be identified by a fluorescent dye having a different wavelength from the fluorescent dye added to the test sample, and base sequences of DNA included in a test sample can be specified by detecting which probe is hybridized with the DNA of the test sample.
The flow path 2a is a portion where a solution including a test sample flows. An inflow port 2b is formed in a concave shape in the upstream side of the flow path 2a, and an outflow port 2c is formed in a concave shape in the downstream side of the flow path 2a. From the inflow port 2b, a solution including a test sample, such as DNA collected from the human body, is injected to flow inside the flow path 2a. At this time, if the test sample includes DNA which is complementary to the probe fixed on the test particle 3, the probe and the DNA are hybridized with each other to be fixed. When the fluorescent intensities of the respective test particles 3 are measured, it can be determined whether the DNA is captured or not.
The method and device for detecting DNA are disclosed in Japanese Unexamined Patent Application Publication No. 2000-346842 (FIGS. 1 and 2).
Even though the diameter of the test particle 3 is about 100 μm, each test particle 3 actually has a tolerance. As shown in
When a test sample DNA is detected by a fluorescence reaction, it is necessary to know which probe is bonded to the test sample DNA. The probe is fixed on the test particle 3, and the test particle 3 is labeled with a fluorescent dye (having a different fluorescence wavelength from a fluorescent dye for labeling sample DNA), so that it can be identified which probe is fixed on a certain test particle. Accordingly, if it is known which of the test particles 3 arranged inside the groove the detected test sample DNA is captured by, the probe to which the sample DNA has complementary sequences is known.
However, although the fluorescence intensities of the test particles 3 are measured one by one by the fluorescence detecting device, tolerances are accumulated as described above, because each test particle has a tolerance. At the present moment, even though the device recognizes that the X-th test particle 3 is tested, it is likely that the test particle 3 which has already been tested is tested, or the test particle 3 which is not tested is skipped so that another test particle 3 adjacent thereto is tested.
In addition, it is preferable that the positions and insertion orders of the test particles 3 inside the flow path 2a be not changed at the time of testing. For example, if the plate substrate 2 is waved up and down to simulate the reaction to a sample DNA, the test particles 3 move to simply change their positions, because there is no place for regulating the movement of the test particles 3 inside the long flow path 2a. Particularly, if the test particles 3 having a small diameter exist, the test particles 3 move over the small tests particles 3 when moving inside the flow path 2a. Therefore, there is a problem in that the insertion order of the test particles is upset.
As a result, the conventional plate has a problem in that the respective test particles 3 cannot be tested with high accuracy.
The present invention has been finalized in view of the drawbacks inherent in the conventional plate, and it is an object of the present invention to provide a plate and a test method using the same capable of performing a test on test particles filled inside a flow path with high accuracy.
According to an aspect of the present invention, a plate includes a flow path having a concave shape and regulation sections that divide the flow path into a plurality of regions extending an upstream side to a downstream side and regulate the number of test particles to be filled inside the regions.
As described above, in the above-mentioned structure, the flow path is divided into a plurality of regions by the regulation sections, and the test particles are filled inside the respective regions. According to this structure, the test particles can be properly positioned in the respective regions.
Further, in the present invention, the flow path is divided into a plurality of regions by the regulation sections, and the test particles filled in each of the regions can be tested in each of the regions. As such, since a test target region can be previously divided into a plurality of regions, the accumulated tolerances of the test particles in the respective regions are small, even though the test particles filled in the respective regions have tolerances. Therefore, the test particles can be tested with high accuracy inside the respective regions.
Furthermore, in the above-mentioned structure, it is preferable that the regions be defined between the regulation sections facing each other in a direction intersecting the flow direction of the flow path. Accordingly, a plurality of regions can be provided easily and properly in the flow path.
Moreover, in the above-mentioned structure, it is preferable that the regulation sections be both side surfaces of the flow path, and that both side surfaces be bent in one or more places from the upstream side to the downstream side. According to this structure, since both side surfaces are bent, regions whose flow directions are different from each other are formed in the upstream side and the downstream side of the bent place, respectively.
More specifically, preferably, the side surfaces of the flow path are composed of first regulation surfaces facing each other in the width direction and second regulation surfaces which are inclined in a different direction from the first regulation surface to face each other in the width direction, and the first regulation surfaces and the second regulation surfaces are alternately disposed from the upstream side to the downstream side in the respective side surfaces. In other words, the flow path is formed in a zigzag shape from the upstream side to the downstream side. Accordingly, the flow path can be divided simply and properly into a plurality of regions for regulating the number of test particles.
Further, in the above-mentioned structure, it is preferable that, inside the flow path, projecting sections provided in one side surface and projecting sections provided in the other side surface are alternately provided from the upstream side to the downstream side to function as the regulation sections.
According to another aspect of the invention, there is provided a test method using the above-described plate. The test method includes filling the test particles into the flow path; setting a test beginning reference position; and testing the test particles filled in each of the regions from the test beginning reference position.
According to this aspect of the invention, the flow path is divided into a plurality of regions by the regulation sections. Then, after test particles are filled inside the respective regions, a predetermined test is performed on the test particles filled inside the respective regions from the beginning reference position. Therefore, the accumulated tolerances of the test particles in the respective regions are small, so that the test particles can be tested with high accuracy in the respective regions.
Further, in the above-mentioned aspect, it is preferable that the beginning reference position be changed whenever a test region is changed. Specifically, it is preferable that a predetermined position of the regulation section for regulating each of the regions be determined to be a test beginning reference position.
As such, whenever the test region is changed, the beginning reference position is also changed. Further, the test particles filled inside the respective regions are tested from a new test beginning reference position, so that the accumulated tolerances of the test particles can be reset in each of the regions, which makes it possible to test the test particles with high accuracy.
A testing plate 1 shown in
The testing plate 1 has a substantially rectangular-parallelepiped shape which has a predetermined thickness to extend in the longitudinal direction (Y1-Y2 direction in
The testing plate 1 is constituted by a plate substrate 12 and a lid body 13. The plate substrate 12 and the lid body 13 are made of, for example, glass or resin. The plate substrate 12 and the lid body 13 are made of a material having a predetermined fluorescence intensity. Particularly, when the testing plate 1 is used as a DNA chip or a protein chip, it is preferable that the testing plate 1 be made of a material, such as silica glass, polydimethylsiloxane (PDMS), or polymethyl methacrylate (PMMA), which exhibits low fluorescence of a substantially transparent color, and has high chemical resistance.
When the testing plate 1 is made of resin, it is preferable that the testing plate 1 be formed by injection molding. In some cases, the testing plate 1 is subjected to hot pressing, so that a groove section to be formed on a top surface 12a of the plate substrate 12 of the testing plate 1 is formed to have a high aspect ratio. In addition, when the testing plate 1 is made of glass, it is molded by hot pressing.
On the top surface 12a of the plate substrate 12 shown in
As shown in
As shown in
As shown in
As shown in
As shown in
Conventionally, a flow path has been formed in a long straight line, as shown in
Accordingly, both sides 14a and 14b of the flow path 14 divide the flow path 14 into the plurality of regions 22 and 23 across the upstream side to the downstream side so as to function as the regulation surfaces 20 and 21 which regulate the number of the test particles 30 to be filled inside the regions 22 and 23.
When the regulation surfaces 20 and 21 are formed in such a manner, the following effects can be expected.
In the related art, a plurality of test particles filled in a flow path having a long straight line are continuously tested one by one from the upstream side to the downstream side (or in the reverse direction). Therefore, if the test particles 30 have tolerances, and when the tolerances are accumulated, the positions of the respective test particles 30 can not be accurately grasped, as the testing reaches the final stage. For example, there is a problem in that the tested particles 30 are tested again. In the present invention, however, the flow path 14 is divided into the plurality of regions 22 and 23 by the regulation surfaces 20 and 21, and the number of the test particles 30 to be filled in the regions 22 and 23 can be regulated. Further, a test is performed on each of the regions 22 and 23, so that the accumulated amount of tolerances of the test particles 30 in the respective regions 22 and 23 is small, which makes it possible to test the test particles 30 with high accuracy.
As shown in
However, in the present invention, after the test particle C is tested, a region to be tested changes from the region 22 to the region 23. Therefore, when the test particles filled inside the region 23 are tested, the accumulated amount of tolerances can be easily reset once here. In other words, the accumulated amount of tolerances is completed in each of the regions 22 and 23, so that the accumulated tolerances cannot be imported when the test particles of the next region are tested, which makes it possible to test the respective test particles 30 with high accuracy.
As such, since the accumulated tolerances of the test particles 30 can be reset in each of the regions 22 and 23, the test particles 30 may be formed to have a relatively rough size, compared to the related art. In other words, as the test particles 30 are formed to have rough sizes, the tolerance thereof becomes large. However, the accumulated tolerances can be reset to the number of the test particles 30 filled in each of the regions 22 and 23, so that a reduction in testing accuracy can be properly controlled.
As shown in
However, in order that the test particles can be continuously inserted from the inflow port 15, inclination angles θ1 and θ2 of the first and second regulation surfaces 20 and 21 with respect to the X2 direction play an important roll. For example, if any one of the inclination angles θ1 and θ2 is more than 90°, there exists a place where the flow direction inside the flow path 14 from the upstream side toward the downstream side faces in the direction parallel to the X1-X2 direction, or where the particles flow reversely from the downstream side (the Y2 side of
As shown in the embodiment of
For example, after the plate 1 is inclined so that the downstream side (the Y2 side of
Moreover, it is preferable that all the regulation surfaces 20 and 21 be surfaces extending in a straight line. Accordingly, two regulation surfaces 20 and 21 are formed in a straight line to intersect at a predetermined angle θ3 in the place where the flow path 14 is bent, so that the filled positions of the test particles 30 are easily determined. In addition, if the angle θ3 to be defined by the regulation surfaces 20 and 21 is very large, the change of flow inside the flow path 14 is too limited. If the testing plate 1 is waved to stimulate mixing, it is highly likely that the test particles 30 move so that their filled positions change. Therefore, it is preferable that the angle θ3 be in the range of 25° to 75°.
A flow path 14 formed in the testing plate 1 shown in
In the embodiment shown in
As shown in
In the embodiment shown in
Similar to the embodiment shown in
In
In the embodiment of
In
For this reason, the test particles 30 are properly stored in the storage portions 84, and the test particles 30 can be prevented from flowing inside the connection path 85 by the regulation surfaces 80 to 83.
In the embodiment shown in
In the embodiment shown in
Hereinafter, a test method according to the present invention will be described, based on the testing plate 1 shown in
In the testing plate 1 shown in
The plurality of test particles 30 are continuously injected through the opening 13a shown in
The test particle 30 is provided with a probe which is attached thereon to capture a specific test sample. The probe which captures a specific test sample is a DNA fragment having a complementary sequence, for example, when the test sample is DNA or RNA. Further, the probe is an antibody to be specifically adsorbed when the test sample is protein. Alternatively, by using the theory of chromatography, test particles can be formed to detect ionic molecules or sugar chain.
The test particles 30 contain or are coated with fluorescent dyes.
As shown in
For example, the probes can be identified by the fluorescent dyes having different wavelengths from the fluorescent dye added to the test sample. By detecting which probe is hybridized with the sample DNA, the base sequence of the DNA included in the test sample can be specified.
The fluorescent intensity can be measured by, for example, a small-sized CCD camera (detecting unit) 28 shown in
A fluorescence detecting device shown in
A laser beam 24 emitted from a laser source 34 is radiated onto the test particles 30 on the plate substrate 12 through a mirror 25 and a lens 26, so that the fluorescent dye in the test particle 30 or the fluorescent dye for labeling a test sample is excited. The fluorescent dye for labeling a test sample is bonded to the test sample which is hybridized with the probe.
Fluorescent light R having a wavelength unique to a fluorescent dye is emitted from the excited fluorescent dye so as to be detected by the CCD camera 28 through the lens 26, the mirror 25, and a filter 27. The mirror 25 and the lens 26 are fixed to a moving plate (moving unit) 29. The moving plate 29 moves in the horizontal direction at the same speed as a cam 35 and a delivering member 31 rotate. As the moving plate 29, the mirror 25, and the lens 26 move in the horizontal direction, the laser beam 24 is sequentially scanned to the test particles 30 arranged inside the flow path 14. Moreover, in the present embodiment, the mirror 25 and the lens 26 of the testing unit are moved in the direction parallel to the flow path 14 by the moving plate 29. However, the testing plate 1 may be moved in the direction parallel to the flow path 14.
In the fluorescence detecting device of the present invention, a control unit 32 for setting a reference position at the beginning of testing is connected to the CCD camera 28. For example, the control unit 32 detects coordinates (X3, Y3) of the first regulation surface 20 shown in
The detection of the reference position at the beginning of testing is performed by detecting a difference in the fluorescent wavelengths. In addition, a calculating unit 33 is connected to the control unit 32 to calculate the amount of movement on the basis of a length L of each of the regions 22 and 23 and the average diameter of the filled test particles 30.
When the test sample DNA captured by the probe is detected by the fluorescent reaction, it is necessary to know which probe is bonded to the test sample DNA. For example, by labeling the test particles with a fluorescent dye (having different wavelengths from the fluorescent dye for labeling the test sample DNA), the test particles 30 can be identified, so that the kind of probe fixed to the test particle 30 can be identified. Accordingly, if it is known which test particle 30 among the test particles 30 arranged inside the flow path 14 captures the detected test sample DNA, a certain probe to which the test sample DNA has complementary sequences is known.
For this reason, when the laser beam 24 is sequentially scanned to the test particles 30, it always needs to grasp which test particle 30 is scanned.
However, the deviation between the estimated position and the actual position of the test particle 30, which is caused by a variation in the diameter of the test particles 30, more easily occurs as the distance from the reference position at the beginning of testing becomes large. In the present invention, however, the fluorescent intensities of the test particles A, B, and C filled inside the region 22 shown in
By the calculating unit 33 shown in
The calculating unit 33 can calculate how many test particles 30 are filled inside the region 22. However, the test particles A, B, and C are completely tested in the order of A→B→C. Then, in order to grasp whether there are actually any other test particles 30 inside the region 22, the moving plate 29 is moved to coordinates (X4, Y4) corresponding to the end of the region 22 to measure the fluorescent intensity. If there are no test particles 30, the fluorescence detecting device measures the fluorescent intensity emitted from the testing plate 1. Therefore, at the time when the fluorescent intensity of the testing plate 1 is measured, the fluorescence detecting device determines that the test particles 30 filled inside the region 22 have been completely tested.
Next, the control unit 32 detects coordinates (X5, Y5) of the second regulation surface 21, which is shown in
Herein, the moving plate 29 and the laser beam 24 move by a predetermined moving amount from the coordinates (X5, Y5) of the second regulation surface 21 to coordinates (X7, Y7) of the second regulation surface. At this time, however, since the testing of the test particle C has been already completed, the moving plate 29 is moved to coordinates (X7, Y7), and then the test is performed, without performing testing only one time.
Alternatively, the beginning reference position is determined to be coordinates (X6, Y6), which is the border between the test particle C and the test particle D. Then, the test may start.
Various test techniques are considered, but the present invention has a specific feature in which the beginning reference position can be changed whenever the testing regions 22 and 23 are changed. Preferably, the beginning reference position can be easily changed on the basis of the coordinates of the regulation surfaces 20 and 21.
When the test particles 30 filled inside the long flow path 14 according to the related art are continuously tested, the beginning reference position cannot be actually changed, because there is no reference required for changing the beginning reference position. Therefore, as the testing reaches the final stage, the accumulated amount of tolerances of the test particles 30 becomes larger, so that aberration easily occurs in the test.
In the present invention, however, the beginning reference position can be easily changed in each of the regions 22 and 23. Therefore, the accumulated tolerances of the test particles 30 can be reset whenever the testing region is changed, which makes it possible to test the test particles 30 with high accuracy.
As described above, according to the present invention, the flow path is divided into a plurality of regions by regulation sections, and the test particles filled inside each of the regions can be tested in each of the regions. Therefore, even though the test particle has a tolerance, the accumulated tolerances in the respective regions are small, which makes it possible to perform a test with high accuracy.
Number | Date | Country | Kind |
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2004-264892 | Sep 2004 | JP | national |