This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2003-400878, filed Nov. 28, 2003, the entire contents of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates to a nucleic acid detecting cassette for detecting nucleic acid, a nucleic acid detecting device utilizing the nucleic acid detecting cassette and a nucleic acid detecting system utilizing the nucleic acid detecting device, particularly, to a nucleic acid detecting closed cassette adapted to an automatic successive processing throughout the entire procedure of detecting the target nucleic acid including the step of putting a sample containing nucleic acid into the nucleic acid detecting cassette, the amplification of nucleic acid and other required processing, and the detection of the target nucleic acid, as well as to a nucleic acid detecting deice and a nucleic acid detecting system each utilizing the particular nucleic acid detecting cassette.
2. Description of the Related Art
As a result of the advent of the technology for amplifying nucleic acid and the improvement in the technology for detecting nucleic acid, the detection of a specified DNA strand has come to be propagated. However, in the amplification of nucleic acid, it may be possible for the environment to be contaminated by the amplified nucleic acid. Also, since complex operations relating to, for example, the temperature conditions, the injection of the solution, and the mixing of the solutions are required for the detection of nucleic acid, the detection of nucleic acid by the application of these technologies is limited to that for the testing and the research.
Proposed in, for example, U.S. Pat. No. 5,229,297 is a throwaway type closed detection container in which a series of operations starting with the processing of a sample containing nucleic acid and ending with the detection of the target nucleic acid are automatically carried out in succession. Also disclosed is a detecting apparatus using the particular detection container. The detection container and the detecting apparatus disclosed in the U.S. patent document quoted above are intended to overcome the above-noted problems so as to make it possible to detect nucleic acid in, for example, a hospital, a clinical laboratory, and a quarantine office.
To be more specific, disclosed in the U.S. patent document quoted above is a channel structure in which a series of steps starting with the amplification and ending with the inspection of nucleic acid, which are carried out by utilizing a pouch type cuvette, can be carried out continuously. However, the pouch type cuvette is unstable in shape so as to give rise to the problem that an undesirable entry of air bubbles is unavoidable in the injecting stage of the solution.
An object of the present invention is to provide a nucleic acid detecting closed cassette adapted to an automatic successive processing throughout the entire procedure of detecting the target nucleic acid including the amplification of nucleic acid, other required processing, and the detection of the target nucleic acid, and to provide a nucleic acid detecting device and a nucleic acid detecting system each utilizing the particular nucleic acid detecting cassette.
According to an aspect of the present invention, there is provided a nucleic acid detecting cassette in which a channel is formed by the combination of a stationary member and a flexible member for detecting nucleic acid contained in a sample, wherein:
the cassette comprises:
a fluid holding channel capable of varying the inner volume;
an inlet-outlet port connected to the fluid holding channel and capable of selecting an open-state under which the communication with the outer portion of the nucleic acid detecting cassette can be achieved and a closed-state under which the communication with outside of the nucleic acid detecting cassette can be interrupted;
a joining channel connected to the fluid holding channel and capable of selecting an open-state under which the fluid transfer to the fluid holding channel can be achieved and a closed-state under which fluid transfer to the fluid holding channel can be interrupted;
an inlet-outlet opening-closing means capable of maintaining the inlet-outlet port under the closed-state; and
a joining channel opening-closing means capable of maintaining the joining channel under the closed-state.
According to another aspect of the present invention, there is provided a nucleic acid detecting device, comprising the nucleic acid detecting cassette defined above, all necessary reagents loaded in the fluid holding channels of the nucleic acid detecting cassette, and a nucleic acid probe of a single stranded nucleic acid immobilized within the detecting channel of the nucleic acid detecting cassette and having a base sequence complementary to that of nucleic acid to be detected.
Further, according to still another aspect of the present invention, there is provided a nucleic acid detecting system, comprising:
the nucleic acid detecting device defined above;
a device holding means for holding the nucleic acid detecting device;
a first driving mechanism for deforming the flexible member against a first region of the fluid holding channel within the nucleic acid detecting device held by the device holding means so as to deform the fluid holding channel;
a second driving mechanism for deforming the flexible member against a second region of the fluid holding channel within the nucleic acid detecting device held by the device holding means so as to deform the fluid holding channel;
a joining channel opening-closing driving mechanism for driving the joining channel opening-closing means; and
a temperature control means for controlling the temperature of the fluid holding channel and the detecting channel.
The present invention relating to the nucleic acid detecting device and the nucleic acid detecting system noted above also constitutes an invention of the method utilizing the nucleic acid detecting device and the nucleic acid detecting system noted above.
The nucleic acid detecting cassettes for detecting nucleic acid according to embodiments of the present invention, the nucleic acid detecting device utilizing the particular nucleic acid detecting cassette and the nucleic acid detecting system utilizing the particular nucleic acid detecting device will now be described with reference to the accompanying drawings.
(1) Basic Construction of Cassette:
The basic construction of the nucleic acid detecting cassette for detecting nucleic acid will now be described first with reference to
(1)-1 Construction of Detecting Cassette:
(1)-2 Material of Detecting Cassette:
The flexible sheet 2 is formed of a flexible member and is arranged to cover the upper surface of the channel formed by the stationary base plate 1. Also, the flexible sheet 2 is sandwiched between the cover plate 3 and the stationary base plate 1, and the cover plate 3 is provided with a pushing block 4 for locally pushing and deforming the flexible sheet 2.
The stationary base plate 1 is formed of a polymeric material such as polypropylene, polycarbonate, POM, or PMMA; silicon; glass; ceramic materials; or a metal such as stainless steel, or aluminum. The flexible sheet is formed of a high molecular weight elastomer such as a silicone rubber, a polypropylene rubber or a urethane rubber. Further, the cover plate 3 is formed of a polymeric material such as polypropylene, polycarbonate, POM, or PMMA; or a metal such as silicon, stainless steel or aluminum. Where each of the stationary base plate 1, the flexible sheet 2 and the cover plate 3 is formed of a plurality of parts, it is possible for the materials described above and/or other materials to be selected and combined appropriately so as to form the parts noted above.
(1)-3 Block Structure of Detecting Cassette:
The nucleic acid detecting cassette 100 is separated into a plurality of blocks having the functions described herein later imparted thereto. In the example of the construction shown in
A nucleic acid detecting chip 500 for detecting nucleic acid is held stationary on the lower surface of the detecting section block 106.
A detecting channel seal 520 is sandwiched between a nucleic acid detecting chip 500 and the lower surface of the detecting section block 106 in a manner to form the channel for the detection. Also, two contact point openings 151 extending from the front surface to reach the back surface are formed in the detecting section block 106. An electric connector (not shown) is inserted into the contact point opening 151 so as to be brought into contact with the exposed surface of the nucleic acid detecting chip 500, with the result that an electric signal generated from the chip surface is output from the electric connector.
(2) Channel and Pushing Mechanism:
The construction that permits the pushing block 4 to deform the flexible sheet 2 so as to vary the inner volumes in the desired portions of the channel and the operation of the particular construction will now be described with reference to
(2)-1 Intermediate Section Block 101:
(2)-2 Edge Section Blocks 102, 103 and Detecting Section Block 106
Like the intermediate section block 101, the edge section block 102 is provided with a substantially rectangular fluid holding channel 111 for holding the fluid. Also, inlet-outlet channels 114 and 115 are connected, respectively, to a channel starting edge section and a channel terminating edge section of the fluid holding channel 111, and a joining channel 117 is further connected to the channel terminating section. Unlike the intermediate section block 101, the edge section block 102 is not provided at the channel starting edge section with a joining channel that is joined to the channel of another block.
The detecting section block 106 is provided with a substantially rectangular retreating channel 131 for retreating the fluid in place of the fluid holding channel 111. The retreating channel 131 has a construction substantially equal to that of the fluid holding channel 111. Joining channels 116 and 117 are connected, respectively, to the channel starting edge section and the channel terminating edge section of the retreating channel 131, and an inlet-outlet channel is not formed in the retreating channel 131. Also, a channel 118 is further connected to the joining channel 116, and a channel 119 is further connected to the joining channel 117. These channels 118 and 119 are connected, respectively, to a left side guide hole 126 (not shown) and a right side guide hole 127 (not shown). A left side guide hole 126 and a right side guide hole 127 are formed to extend to reach the back surface of the detecting section block 106). The detecting channel formed on the back surface of the detecting section block 106 is connected to the channels 118 and 119 via the left side guide hole 126 and the right side guide hole 127 noted above. It follows that the fluid is allowed to be transferred between the retreating channel 131 and the detecting channel.
Like the intermediate section block 101, the edge section block 103 is provided with a substantially rectangular fluid holding channel 111 for holding the fluid. Inlet-outlet channels 114 and 115 are connected, respectively, to the channel starting edge section and channel terminating edge section of the fluid holding channel 111. Further, the joining channel 116 is connected to the channel starting edge section noted above. However, unlike the intermediate section block 101, the edge section block 103 is not provided with a joining channel at the channel terminating edge section.
(2)-3 Main Dimensions and Shapes of Channel:
The dimensions of each of the constituents of the blocks shown in
Each of the fluid holding channel 111 and the retreating channel 131 has a depth of about 0.5 mm, a length toward the adjacent channel, i.e., the length between the channel starting edge section and the channel terminating edge section, of about 10 mm, a length in a direction perpendicular to the direction toward the adjacent channel, i.e., the width of the channel, of about 10 mm, and a standard holding inner volume of about 40 μl (micro-liters) to 48 μl. In the construction that the pushing block 4 opens outward, the flexible sheet 2 is capable of expansion outward, with the result that it is possible for each of the fluid holding channel 111 and the retreating channel 131 to keep a holding inner volume that is about 2 to 3 times as large as the standard holding inner volume noted above. Incidentally, the retreating channel 131 is kept shrunk before initiation of the nucleic acid detecting operation so as to have a small inner volume, and the inner volume of the retreating channel 131 can be expanded at the initiating stage of the detecting operation. It should be noted that the difference in the inner volume of the retreating channel 131 between the expanded stage and the shrunk stage is set to a level not smaller than the volume of the fluid loaded in the detecting channel.
Each of the inlet-outlet channels 114 and 115 has a depth of about 0.25 mm, a length toward the fluid holding channel of about 2 to 3 mm, and a length in a direction perpendicular to the direction toward the fluid holding channel, i.e., the width of the channel, of about 2 mm. It is possible to set each of the inlet-outlet channels 114 and 115 in a manner to form a completely closed state such that the inner volume of each of the inlet-outlet channels 114 and 115 is substantially zero.
Each of the joining channels 116 and 117 has a depth of about 0.25 mm, a length toward the adjacent channel of about 2 to 3 mm, and a length in a direction perpendicular to the direction toward the fluid holding channel, i.e., the width of the channel, of about 2 mm. It is possible to set each of the joining channels 116 and 117 in a manner to form a completely closed state such that the inner volume of each of the joining channels 116 and 117 is substantially zero.
It should be noted that the walls defining the channel, which extend from the bottom surface to the side surface of the channel, are smoothly curved so as to make it possible to set up the state that, even when the flexible sheet 2 is flexed, an internal overstress is not generated and the flexible sheet 2 can be brought into a tight contact without fail with the bottom surface of the channel such that the inner volume of the channel can be made substantially zero.
The flexible sheet 2 has a thickness of 0.2 to 0.5 mm, and can be formed of a relatively hard material having a rubber hardness of JIS-A20° to 30° or a relatively soft material having a rubber hardness of Asker C20° to 40°.
(2)-4 Pushing Pad:
A central pad 401, a left side pad 402, and a right side pad 403 are arranged on that region of the flexible sheet 2 which is positioned to face the fluid holding channel 111. The left side pad 402 and the right side pad 403 are arranged, respectively, on the upper portions of the channel starting edge section and the channel terminating edge section of the fluid holding channel 111 and in the vicinity of the upper portions noted above. On the other hand, the central pad 401 is arranged to face that region of the fluid holding channel 111 in which the width of the channel is broadened. In addition, the central pad 401 is positioned in contact with the left side pad 402 and the right side pad 403. The upper portion of the fluid holding channel 111 is substantially covered with these pads 401, 402 and 403. In the example of the construction shown in
A left side inlet-outlet pad 404 and a right side inlet-outlet pad 405 are arranged, respectively, above the upper portions of the inlet-outlet channels 114 and 115.
Also, a left side joining pad 406 and a right side joining pad 407 are arranged on the upper portions of the joining channels 116 and 117, respectively.
Each of these pads is shaped to permit the flexible sheet 2 to be brought into a tight contact with the corresponding channel formed in the stationary base plate 1 so as to decrease the inner volume of the channel to substantially zero when the flexible sheet 2 is pushed substantially in the vertical direction from the front surface toward the stationary base plate 1.
(2)-5 Channel Cross Sectional Mechanism:
As shown in
As shown in
Needless to say, the inner volume of the retreating channel 131 can also be varied similarly like the fluid holding channel 111 described previously with reference to
(2)-6 Opening-Closing Mechanism of Pushing Block 4:
(2)-6-1 Basic Structure of Pushing Block:
The related motions of the parts of the pushing block 4 and the cover plate 3 shown in
Various methods can be applied for the movement and fixation of the pushing pads 401 to 408. In the nucleic acid detecting cassette according to the first embodiment of the present invention, employed is the construction comprising a movable rod having a single fulcrum and a locking section for temporarily fixing the edge section of the movable rod, as described in the following.
As shown in
The movable rods 411 to 417 can be fixed by mainly two methods. In one of the two methods, a claw-shaped member for maintaining the pushed and fixed state is fitted in a concave portion formed in the locking section so as to push down and fix the rod. The movable rods 411 to 413, 416 and 417 are fixed by this fixing method. In the other fixing method, the pushed and fixed state is maintained by inserting the locking key into the region between the rod and the cover plate 3 so as to upheave and fix the rod. The movable rods 414 and 415 are upheaved and fixed by this fixing method.
In the movable rod that is fixed by the pushing and fixing method, a fulcrum section, a rod-like member, a claw-shaped member and a pad are formed integral. Also, the locking section comprises a claw-shaped member and a concave section. As shown in
The fulcrum holes 451, 461, 471, 481, and 491 are movably mounted, respectively, to a series of rear fulcrum holes 446 fixed to the cover plate 3. To be more specific, the movable rods 411 to 413, 416 and 417 are rotatably supported within a movable range such that the movable rods 411 to 413, 416 and 417 are rendered rotatable about the fulcrum holes 451, 461, 471, 481 and 491, respectively, by allowing a fulcrum bar to extend through each of the holes of the rear fulcrum holes 446 and each of the holes of the fulcrum holes 451, 461, 471, 481 and 491.
It is possible for the locking sections 431, 432, 433, 436 and 437 formed in the cover plate 3 to permit the movable rods 411 to 413, 416 and 417 to maintain the closed state of the corresponding channel portion. To be more specific, if the claw-shaped members 453, 463, 473, 483 and 493 formed in the rods 411 to 413, 416 and 417, respectively, are pushed downward from the outside so as to be fitted in and fixed within the concave sections of the locking sections 431, 432, 433, 436 and 437, the rods 411 to 413, 416 and 417 are held under the pushed down state so as to close the channels corresponding to the pads 401, 402, 403, 406 and 407.
The portions of the claw-shaped members 453, 463, 473, 483 and 493 of the locking sections 431, 432, 433, 436 and 327 are elastically flexed upon receipt of a driving force applied from the outside so as to release the fitting of the claw-shaped members 453, 463, 473, 483 and 493 into the locking sections 431, 432, 433, 436 and 327, with the result that the movable rods 411, 412, 413, 416 and 417 are individually opened. If the movable rods 411, 412, 413, 416 and 417 are opened, the flexible sheet 2 is brought back to the original stage before the flexing by the resilience of the flexible sheet 2 itself so as to open the corresponding channel portion. It should be noted, however, that the opening force of the movable rods 411, 412, 413, 416 and 417 is braked by the driving force applied from the outside so as to make it possible to control the opening speed of the movable rods noted above.
Incidentally, each of the channels of the retreating channel 131 can be closed and opened by the similar mechanism.
The movable rod that can be fixed by the upheaving and fixing method is constructed to include a rod-like member, a fulcrum and a pad which are formed integral. Also, the locking key includes a wedge section. The inlet-outlet pads 404 and 405 are fixed, respectively, to the one-side edges of the rod-like members 414a and 415a of the movable rods 414 and 415. Also, fulcrum holes 414b and 415b are mounted, respectively, to the intermediate portions of the rod-like members 414a and 415a.
The fulcrum holes 414b and 415b are rotatably mounted to a series of forward fulcrum holes 444a and 444b, respectively, which are fixed to the cover plate 3. To be more specific, a fulcrum bar (not shown) is allowed to extend through each of the holes of the forward fulcrum holes 444a, 44b and each of the holes of the fulcrum holes 414b and 415b so as to permit the movable rods 414 and 415 to be made rotatable within a rotatable range about the fulcrum holes 414b and 415b, respectively.
The locking keys 434 and 435 arranged on the cover plate 3 permit the movable rods 414 and 415, respectively, to maintain the closed state of the inlet-outlet channels 114 and 115. To be more specific, if the locking key 434 is inserted into the regions between the movable rod 414 and the cover plate 3 so as to upheave and fix the movable rod 414, the inlet-outlet channel 114 is closed by the movable rod 414. The locking keys 434 is withdrawn from the regions between the movable rod 414 and the cover plate 3 by the driving force applied from outside the nucleic acid detecting cassette 100, with the result that the movable rod 414 is opened and, thus, the inlet-outlet channel 114 is opened. In this case, the flexible sheet 2 is brought back to the original state by the resilience of the flexible sheet 2 itself. The closed state of the inlet-outlet channel 115 can also be maintained and opened with the movable rods 415 and the locking keys 435 by the similar mechanism.
(2)-6-2 Fluid Holding Channel 111 and Retreating Channel 131:
The opening-closing operations of the fluid holding channel 111 and the retreating channel 131 will now be described in detail with reference to
Each of the fluid holding channel 111 and the retreating channel 131 is capable of realizing various inner volume patterns of the channel by individually driving the central rod 411, the left side rod 412 and the right side rod 413.
Examples of various patterns ranging between the completely closed state and the completely opened state of the fluid holding channel 111 will now be described with reference to
As shown in
The description given above is directed to the opened state and the closed state of the fluid holding channel 111. Since the retreating channel 131 is also opened or closed like the fluid holding channel 111, the description is omitted in respect of the opening and the closing of the retreating channel 131.
(2)-6-3 Joining Channels 116 and 117:
The opening-closing operations of the joining channels 116 and 117 will now be described with reference to
Incidentally, the opening-closing of the joining channel 117 is also controlled like the joining channel 116 and, thus, the description is omitted in respect of the joining channel 117.
(2)-6-4 Inlet-Outlet Channels 114 and 115:
The opening-closing operation of the inlet-outlet channels 114 and 115 will now be described with reference to
Also, if the locking key 434 is withdrawn by an external driving force so as to release the movable rod 414 from the locking key 434 as shown in
Incidentally, the opening-closing of the inlet-outlet channel 115 can also be controlled like the inlet-outlet channel 114 and, thus, the description in conjunction with the inlet-outlet channel 115 is omitted.
(3) Method of Controlling Fluid Movement:
The control method of the fluid moving within the channel will now be described. Used in this control method is the pushing mechanism described above, which permits varying the channel and the inner volume thereof.
(3)-1 Injection of Reagent and Sample:
(3)-1-1 Injection of Reagent Solution:
The reagent is injected into the fluid holding channel 111 by utilizing the opening portions 121 and 122 formed in the back surface of the nucleic acid detecting cassette 100, i.e., inlet-outlet port. The process of injecting the reagent solution into the fluid holding channel 111 will now be described with reference to
Specifically,
On the other hand, the valve mechanisms achieved by the combination of the joining channels 116, 117, the joining pads 406, 407 and the flexible sheet 2 are equivalently represented by joining valves 316 and 317 in
Concerning the fluid holding channel 111 capable of varying the inner volume, the planar shape of the inner volume is schematically shown in
a. Setting of Initial Inner Volume of Channel:
In the first step, the inlet-outlet valves 314 and 315 are opened with the joining valves 316 and 317 kept closed as shown in
b. Start-Up of Reagent Injection:
If the initial inner volume of the fluid holding channel 111 is determined in the process (a) given above, i.e., the process of setting the initial inner volume of the channel, the injection of the reagent solution is started.
In the next step, the reagent 303 is injected from the tip 301 as shown in
c. Termination in Injection of Reagent Solution:
The reagent solution 303 is injected until the reagent solution 303 flows into the tip 302 on the outlet side of the gas, as shown in
d. Injection of Replenishing Reagent Solution:
As shown in
The reagent solution is further injected until the reagent 303 remains slightly within the tip 301 on the inlet side of the reagent solution, and the reagent 303 is pushed in until the inner region of the fluid holding channel 111 is slightly pressurized. Then, the pressure within the opening portion of the tip 301 and the pressure inside the fluid holding channel 111 are rendered substantially equal to the atmospheric pressure so as to confirm that the amount of the reagent solution 303 within the tip 301 is slightly increased. In this case, if the reagent 303 remains inside the tip 301, the gas is not involved in the reagent solution, even if the pressure inside the fluid holding channel is negative so as to cause the reagent 303 within the tip 301 to be slightly sucked into the fluid holding channel 111.
e. Closure of Inlet-Outlet Valve:
Finally, the inlet-outlet valves 314 and 315 are closed, and the tips 301 and 302 are detached from the opening portions 121 and 122, respectively, as shown in
By the process steps described above, it is possible to inject the reagent solution into the fluid holding channel 111 under the two-stage of set amounts while preventing the gas from being involved in the reagent solution as air bubbles. In the stage of injecting the reagent solution, the pressure inside the flexible sheet 2 is equal to the pressure outside the flexible sheet 2, with the result that the flexible sheet 2 is allowed to retain a prescribed shape so as to maintain constant the loaded amount of the reagent solution. Further, since an extremely high negative pressure or positive pressure is not applied to the reagent solution, the gas is unlikely to enter the fluid holding channel 111 from the outside or the reagent solution is unlikely to leak from within the channel. In addition, since a gaseous portion is not included inside the channel, it is possible to prevent the gas from being dissolved in the reagent solution during the storage over a long period of time.
(3)-1-2 Injection of Sample:
The method of injecting a liquid sample 305 containing a nucleic acid material, which is to be newly inspected, into the fluid holding channel 111 loaded in advance with a prescribed amount of the reagent solution will now be described with reference to
a. Initiation of Sample Injection:
As shown in
In the next step, the nucleic acid detecting cassette 100 is held such that the opening portions 121 and 122 are positioned on the upper side. Under this condition, the tip 301 of the pipette 300 loaded with the sample 305 to be injected is inserted into the opening portion 121. At the same time, the tip 302 open to the outside is inserted into the opening portion 122 on the opposite side of the opening portion 121. By the mounting of these tips 301 and 302, the setting of the cassette is finished, and the injection of the sample 305 from the tip 301 is started.
b. Termination of Sample Injection:
As shown in
c. Closure of Inlet-Outlet Valve:
Finally, the inlet-outlet valves 314 and 315 are closed, and the tips 301 and 302 are detached from the opening portions 121 and 122, respectively, as shown in
Where the sample 305 has a specific gravity larger than that of the reagent 303 and also has a diffusion coefficient smaller than that of the reagent 303, and where the direction of the gravity of the sample 305 is on the side opposite to the side of the inlet-outlet valves 314 and 315 as viewed from the center of the fluid holding channel 111 as denoted by an arrow in
By the process steps described above, it is possible to inject the nucleic acid material to be inspected into the fluid holding channel 111 that is loaded in advance with a prescribed amount of the reagent while preventing the gas from being involved in the sample and the reagent as air bubbles.
(3)-2 Fluid Transfer:
The method of transferring the fluid among a plurality of fluid holding channels 111 having the reagent injected thereinto by the fluid injecting process will now be described. The fluid transfer method described in the following covers a case where a prescribed amount of the fluid is transferred and another case where the maximum holding amount of the fluid is transferred. Also, the following description covers the case where the fluid is transferred between the fluid holding channel 111a and the fluid holding channel 111b.
(3)-2-1 Transfer of Prescribed Amount of Fluid:
In the transfer of a prescribed amount of the fluid, a prescribed amount of the fluid is transferred from the fluid holding channel 111a holding the maximum amount of the fluid into the fluid holding channel 111b holding the minimum amount of the fluid so as to increase the amount of the fluid held in the fluid holding channel 111b to reach the maximum holding amount. The minimum holding amount denotes the inner volume in the case where the left side pad 402 and the right side pad 403 shown in
The process of transferring a prescribed amount of the fluid will now be described in detail.
a. Opening of Joining Valve:
As shown in
In the next step, the central pad 401a of the fluid holding channel 111a is slightly pushed in so as to pressurize the fluid such that the joining valve 317 is completely opened. It should be noted that, before the pressurizing step, the flexible sheet 2 constituting the joining valve 317 is pushed against the bottom surface of the joining channel 117 in a manner to eliminate completely the clearance between the flexible sheet 2 and the bottom surface of the joining channel 117. It follows that, even if the locking section 437 of the movable rod 417 for locking the joining channel 117 is released, the flexible sheet 2 in that portion is pushed against the bottom surface by the atmospheric pressure in the case where the resilience of the flexible sheet 2 is weak. Such being the situation, it is possible for the joining valve 317 to fail to be opened completely. However, a restoring force is imparted to the flexible sheet 2 by pressurizing the fluid holding channel 111a so as to cause the joining valve 317 to be opened without fail.
b. Start-Up of Fluid Transfer:
As shown in
In this case, it is possible to start the fluid transfer such that the pressure of the fluid holding channel 111b is rendered equal to the pressure of the fluid holding channel 111a before the central pad 401a of the fluid holding channel 111a is pushed in.
It is possible to put the central pad 401b of the fluid holding channel 111b under the opened state before the joining channel 117 is opened. In this case, however, a negative pressure is set up within the fluid holding channel 111b, with the result that it is possible for the joining channel 117 to fail to be opened smoothly.
c. Termination of Fluid Transfer and Closure of Joining Valve:
As shown in
In the process of transferring a prescribed amount of the fluid described above, the volume Vt of the fluid that is transferred meets approximately the relationship of Vt=Vmax−Vmin, where Vmax denotes the maximum holding amount of the fluid holding channel, and Vmin denotes the minimum holding amount of the fluid holding channel, in the case where the locking mechanism of the fluid holding channel is of a single stage structure.
(3)-2-2 Fluid Transfer in Maximum Holding Amount:
In the fluid transfer operation in the maximum holding amount, a fluid in the maximum holding amount is transferred from the fluid holding channel 111a holding the maximum holding amount to the fluid holding channel 111b under the completely closed state, and the amount of the fluid in the fluid holding channel 111b is set at the maximum holding amount of the fluid holding channel.
The inner volume under the completely closed state corresponds to the inner volume under the state that all of the central pad 401, the left side pad 402 and the right side pad 403 are closed. Unless the rubber hardness of the flexible sheet 2 is large and unless the seams between the fluid holding channel 111 and each of the inlet-outlet channels 114, 115, and the joining channels 116, 117 are processed smoothly, it is possible for a clearance to be generated within the fluid holding channel 111 even under the completely closed state. However, since a liquid material or fluid in an amount intermediate between the minimum holding amount and the maximum holding amount is injected initially into the fluid holding channel 111, the clearance noted above is filled with a residual liquid material or a residual fluid, with the result that it is impossible for air bubbles to be contained in the fluid holding channel 111.
The process of transferring the fluid in the maximum holding amount will now be described.
a. Opening of Joining Valve:
As shown in
In the next step, the central pad 401a of the fluid holding channel 111a is slightly pushed in so as to achieve the pressurization such that the joining valve 317 is completely opened.
b. Start-Up of Fluid Transfer:
As shown in
c. Intermediate Stage of Fluid Transfer:
As shown in
d. Termination of Fluid Transfer and Closure of Joining Valve:
As shown in
It should be noted that the maximum holding amount Vmax of the fluid holding channel 111b meets the relationship of Vmax=Vi−Vr, where Vi denotes the total inflow amount into the fluid holding channel, and Vr denotes the amount of the residue in the fluid holding channel.
(3)-2-3 Effects of Fluid Transfer:
The fluid transfer method described above makes it possible to obtain the effect given below:
a. It is possible to transfer the fluid in the equal volume under a small pressure difference.
The inner volume of each channel is variable. However, the inner volume of the entire channel is substantially constant. Also, the pressure difference required for the fluid transfer is generated by varying the inner volume of the variable-volume channel itself. As a result, it is possible to diminish the pressure difference among the channels and the pressure difference between the inner region of the channel and the outside, compared with the fluid transfer system in which pressure is applied to the both edges of the entire channel. It follows that the sealing of the liquid material and the control of the fluid transfer can be performed without fail.
The fluid transfer method described above also makes it possible to produce the effect given below:
b. It is possible to achieve the related motion fluid transfer.
It is possible to drive the system by related moving the pressurizing pads of the fluid holding channels on the fluid transferring side and on the fluid receiving side. In this case, the pushing velocity of the pressurizing pad on the fluid transferring side is made substantially equal to the releasing velocity of the pressurizing pad on the fluid receiving side. It follows that it is possible to diminish further the pressure difference among the channels and the pressure difference between the inner region of the channel and the outside.
(3)-2-4 Air Bubble-Free Closure of the Joining Channel:
In order to ensure the air bubble-free closure of the joining channel, it is possible to introduce a small amount of the reagent into the joining channel so as to load even a small clearance with the liquid material when the liquid material is injected into the fluid holding channel or before the injecting stage of the liquid material. In this case, it is necessary to use a liquid material that does not give adverse effects to the reactions on the fluid holding channels on both sides of the joining channel.
(3)-3 Mixing:
A plurality of reactions are required in the steps of continuously performing the required processing throughout the entire system including the putting of the sample containing nucleic acid, the nucleic acid amplification and other required treatments, and the detection of the target nucleic acid. In the channel system according to the first embodiment of the present invention, various reactions are consecutively carried out by the steps of, for example:
a. Transferring the reaction product within the preceding fluid holding channel into the succeeding fluid holding channel;
b. Mixing the reagent loaded in advance within the succeeding channel with the reaction product transferred into the succeeding channel; and
c. Transferring a new reaction product into the succeeding fluid holding channel.
In this fashion, it is possible to realize a consecutive processing. The mixing method of the reagent required for the consecutive processing described above will now be classified into (1) the fluid transfer under the pressurized state within a single channel, (2) the isobaric fluid transfer within a single channel, and (3) the reciprocating fluid transfer between two channels, and will now be described in the classified fashion.
(3)-3-1 Fluid Transfer Under Pressurized State within Single Channel:
Under the state that the central pad 401a is under the completely closed state and the joining valve 317 is also under the completely closed state as shown in
As shown in
In this mixing method, the fluid holding channel 111b holds the fluid up to the maximum holding amount. Therefore, if the pushing amounts of the pushing pads 401b, 402b and 403b are large, the internal pressure of the fluid holding channel 111b is increased resulting in the fluid leakage. Such being the situation, it is necessary to control the pushing amounts of the pushing pads 401b, 402b and 403b. The transfer method in which the fluid is pressurized within a single channel corresponding to the fluid holding channel 11b so as to bring about the transfer of the fluid within the single channel as shown in
(3)-3-2 Isobaric Fluid Transfer within Single Channel:
It is possible to increase the transfer amount of the fluid inside the fluid holding channel 111b without excessively increasing the internal pressure of the fluid holding channel. In this case, a sufficiently large amount of the fluid is supplied into the fluid holding channel 111b in the two-stage transfer of the fluid employed in the process of the isobaric transfer within a single channel, which is described herein later, so as to transfer the fluid in an isobaric fashion within the single channel.
a. Initial Fluid Transfer:
As shown in
b. Initial Mixing:
After about 24 μl of the fluid has been transferred, the joining valve 317 is put under the completely closed state. Under this condition, the left side pad 402a, the right side pad 403b, and the central pad 401b are consecutively pushed down in a prescribed amount as shown consecutively in
In the example described above, about 24 μl of the fluid is transferred. Since the maximum holding amount of about 48 μl has a sufficient allowance relative to about 24 μl of the transferred fluid, the inner pressure of the fluid holding channel 111b is not increased to exceed the external pressure even if the depressing amount of the pushing pad is set relatively large. It follows that it is unnecessary to worry about the fluid leakage. Under this state, it is possible to bring about the transfer of the fluid inside the fluid holding channel 111b in an amount that is sufficient for the mixing.
c. Latter Stage Fluid Transfer:
As shown in
d. Latter Stage Mixing:
The latter stage mixing is carried out under the state that about 36 μl of the fluid noted above has been transferred. After the latter stage fluid transfer, the fluid holding channel 111a is under the state equal to the state shown in
In the latter stage mixing, more than half the amount of the fluid is already mixed. As a result, a sufficient mixing can be achieved even if the depressing amount of each pad is not excessively increased.
In the example described above, the operation is controlled by the amount of the transferred fluid that is regulated by the pad. However, it is possible for the pushing pad to be held at an optional position, for the joining valve 317 to be put under the completely closed state, and for the mixing of the fluid to be carried out within the fluid holding channel 111b under an optional transfer amount of the fluid.
(3)-3-3 Reciprocating Fluid Transfer Between Two Channels:
Where a prescribed amount of the fluid is transferred from the fluid holding channel 111a into the fluid holding channel 111b, it is possible in some cases to employ the intermediate stage of allowing the fluid to flow backward from the fluid holding channel 111b into the fluid holding channel 111a. In this case, it is possible for the fluid to be reciprocated between the two channels.
(3)-3-4 Difference in Mixing Ratio:
In the mixing methods of the fluid described above, the case A where it is possible to perform the backward transfer of the fluid between the fluid holding channel 111a and the fluid holding channel 111b differs from the case B where it is impossible to perform the backward transfer of the fluid noted above. To be more specific, the cases A and B given above differ from each other in the mixing ratio between the reagent and the reaction product. For example, the mixing ratio of the reagent:reaction product is B0:A1 in the case B where the backward transfer cannot be performed, and the mixing ratio of the reagent:reaction product is B0:A0 in the case A where the backward transfer can be performed, where A0 denotes the amount of the reaction product within the fluid holding channel 111a, A1 denotes the amount of the reaction product transferred into the fluid holding channel 111b, and B0 denotes the amount of the reagent inside the fluid holding channel 111b.
Also, the case where the reaction product is partly allowed to remain inside the fluid holding channel 111a, from which the fluid is transferred, as a residue that is harmful to the reaction carried out inside the fluid holding channel 111b corresponds to the case where it is impossible to perform the backward transfer of the fluid in this mixing method.
(3)-3-5 Effect of Mixing:
Where a plurality of reactions are carried out successively in the gas-liquid two layer system, it was customary in the past to supply the required reagent every time the reaction was carried out in a single reaction chamber. In the method, the amount of the fluid inside the reaction chamber is increased in the every reactions so that a waste liquid chamber is needed, causing a complex channel system. Also, if the amount of the reagent is decreased, it is difficult to control the transfer of the fluid, with the result that a small amount of the reagent remains particularly in inside the long channel. It follows that the waste of the reagent and the nonuniformity in the transfer amount of the fluid are brought about as problems to be solved.
In the fine channel system, it is difficult to stir the fluid within the channel in mixing the different streams of the fluid. Even in the case of using, for example, a combined channel in which different streams of the fluid are combined, it is said to be difficult to achieve a homogenous mixing.
On the other hand, according to the embodiment described above, a relatively large amount of the reaction product itself is transferred, though the amount of the reagent that is transferred is relatively small. Such being the situation, both the transfer and the mixing of the fluid can be controlled easily.
It should also be noted that, in mixing the fluid, it is possible to employ various patterns conforming with the fluid and with the conditions of the reaction. Further, the mixing can be performed under the air bubble-free state. It follows that it is unnecessary to employ a centrifugal separating apparatus for separating the fluid into the gas-liquid two layers.
(3)-4 Modifications:
It is possible to carry out the transfer and mixing of the fluid under more kinds of the volume of the fluid that is held, more kinds of the transfer amount of the fluid, more kinds of the transfer method of the fluid, and more kinds of the mixing method of the fluid by dividing the pushing pad into a larger number of sections and/or forming the locking system for fixing the pushing pad in a manner to have a multi-stage structure in the steps of injecting, transferring and mixing the reagent and the sample. It follows that it is possible to allow the operation in the present invention to conform easily with various fluids and the reacting conditions.
(4) Fluid Transfer Pattern and Order of Fluid Transfer Steps:
(4)-1 Detecting Channel:
(4)-1-1 Detecting Method of Nucleic Acid:
It is possible to use, for example, a known optical system or electrochemical system as the detecting method of the target nucleic acid in the case of using a nucleic acid probe of a single stranded nucleic acid, which has a base sequence complementary to that of the target nucleic acid that is to be detected and which is immobilized inside the detecting channel.
The electrochemical detecting method disclosed in the registered Japanese Patent No. 2,573,443 can be applied basically to the detecting method according to this embodiment of the present invention. Also, it is possible to employ an optical method by using a light transmitting material for forming the stationary base plate 1.
(4)-1-2 Nucleic Acid Detecting Chip:
It is possible to use a detecting base plate 500a consisting of a stationary member as the nucleic acid detecting chip 500 acting as a detecting sensor. To be more specific, used is a nucleic acid detecting sensor as disclosed in Japanese Patent Disclosure (Kokai) No. 2002-195997. The detecting sensor used in the present invention employs, for example, the same detecting method, and has the same material used, and the same electrode structure, though the construction disclosed as a prior art in the patent document quoted above is employed in the present invention as the construction of the detecting base plate 500a.
Also, a nucleic acid probe 502 of a single stranded nucleic acid having a base sequence complementary to that of the target nucleic acid to be detected is immobilized on the nucleic acid immobilizing electrode 501 by the method disclosed in Japanese Patent Disclosure No. 2002-195997 quoted above.
(4)-1-3 Construction of Detecting Channel:
(4)-1-4 Bonding of Detecting Channel to Base Plate:
A seal recess 125 having a depth substantially equal to the thickness of the detecting channel seal 520, which further extends from the bottom surface of the chip recess 128, is further formed in a part of the chip recess 128. The detecting channel seal 520 is incorporated in contact with the plane of the deepest portion of the seal recess 125. Further, the nucleic acid detecting chip 500 is incorporated in contact with the plane of the deepest portion of the chip recess 128 such that the nucleic acid detecting chip 500 is in contact with the detecting channel seal 520.
Because of the construction described above, formed is a detecting channel 531 isolated from the outside and consisting of the nucleic acid detecting chip 500, the detecting channel seal 520 and the detecting section block 106. It is possible for the nucleic acid detecting chip 500, the detecting channel seal 520 and the detecting section block 106 to be bonded or welded or adhered to each other. Also, the nucleic acid detecting chip 500, the detecting channel seal 520 and the detecting section block 106 are integrally bonded to each other by using a fastening member or a fastening portion so as to form the nucleic acid detecting closed cassette 100.
(4)-1-5 Relationship Between Retreating Channel and Detecting Channel:
The detecting section block 106 shown in
To be more specific, the left side of the detecting channel edge portion 118 is joined to the fluid holding channel 111a via the joining channel 117a, and the right side of the detecting channel edge portion 118 is joined to the retreating channel 131 via the joining channel 116b. These joining channels can be opened or closed by the corresponding joining pads 407a and 406b.
Similarly, the left side of the joining channel edge portion 119 is joined to the retreating channel 131 via the joining channel 117b, and the right side of the joining channel edge portion 119 is joined to the fluid holding channel 111c via the joining channel 116c. These joining channels can be opened or closed by the corresponding joining pads 407b and 406c.
(4)-1-6 Contact Point Opening:
As shown in
(4)-2 Initial Injection:
Like
The method disclosed in the registered Japanese Patent No. 2,573,443 and Japanese Patent Disclosure No. 2002-195997 can be applied in each of the process steps unless otherwise specified and, thus, attentions should be paid to these patent documents in respect of each of the process steps. First of all, described is the initial state under which all the required reagents are injected for making the nucleic acid detecting system ready for delivery to the market. Also disclosed is the reaction process using each of the reagents.
(4)-2-1 Nucleic Acid Amplification:
In the fluid holding channel 811a, reactions such as a polymerase chain reaction (PCR) are carried out from the sample containing nucleic acid so as to amplify nucleic acid within the sample. The amplifying method of nucleic acid is not particularly limited. It is possible to employ various nucleic acid amplifying methods including the method accompanied by the change in temperature such as the polymerase chain reaction (PCR) method and the isothermal amplifying method. It is possible to use, for example, the living body samples such as blood, serum, urine, saliva and a mucous membrane of the mouth as the samples containing nucleic acid. In, for example, the PCR method, a thermal circulation is applied as follows so as to amplify nucleic acid continuously.
a. Heating is applied at 92 to 95° C. for about 10 to 15 seconds so as to denature the double stranded nucleic acid, followed by loosening and separating the double stranded nucleic acid so as to form nucleic acid of a single stranded nucleic acid.
b. Then, nucleic acid is cooled and retained at 55 to 65° C. for about 10 to 15 seconds so as to anneal the primer, thereby allowing the two kinds of the primer to be coupled with the separated single stranded nucleic acid so as to form partially a double stranded nucleic acid.
c. Further, nucleic acid is retained at 70 to 72° C. for about 10 to 15 seconds so as to elongate an additional nucleic acid chain having complementarity with the two kinds of the primer used as the starting point of the nucleic acid synthesis.
For carrying out the PCR method, a buffer solution containing dNTP, primer, polymerase, and other reagents required for the PCR method is injected into the fluid holding channel 811a. It is also possible to add, as required, a reagent effective for eliminating the effect of the substance inhibiting the amplification of nucleic acid to the sample solution. For example, it is possible to add “Ampdirect” manufactured by Shimazu Seikakusho K.K. to the sample solution. The reagent effective for eliminating the effect of the substance inhibiting the amplification of nucleic acid is a reagent that permits taking out nucleic acid directly from blood for carrying out the PCR method. The total amount of the reagents injected into the fluid holding channel 811a is about 48 μl.
(4)-2-2 Producing Single Stranded Nucleic Acid:
In the fluid holding channel 811b, the double stranded nucleic acid, which is amplified in the nucleic acid amplifying process carried out in the fluid holding channel 811a, is produced the single stranded nucleic acid by, for example, λexonuclease method. In this stage, the sample is retained at 35 to 39° C. for about 30 minutes to 3 hours in order to maintain the enzyme reaction. Finally, the sample is heated at 92 to 95° C. for about 3 to 6 minutes for deactivating the enzyme. The total amount of about 12 μl of exonuclease and the reagents contained in the buffer solution, which are used in this stage, are injected into the fluid holding channel 811b.
(4)-2-3 Impartation of Protective Chain:
In the fluid holding channel 811c, the protective chain nucleic acid chain complementary to the sequence of the portion of amplified single stranded nucleic acid sample that is not complementary to the nucleic acid probe 502 is added to the amplified single stranded nucleic acid sample used by the method disclosed in Japanese Patent Disclosure No. 6-70799. As a result, the self-hybridization of the amplified nucleic acid sample can be prevented so as to improve the detection sensitivity. In this stage, the hybridization reaction is carried out, if required, between the protective chain and the nucleic acid sample by retaining the nucleic acid sample at 95 to 98° C. for about 1 to 5 minutes so as to thermally denature nucleic acid, followed by moderately cooling the nucleic acid sample to 25° C. at a cooling rate of 2° C./min. The total amount of about 12 μl of the reagents used in this process are injected into the fluid holding channel 811b.
(4)-2-4 Hybridization:
In the detecting channel 531, the nucleic acid sample that was amplified and pretreated and the nucleic acid probe 502 on the nucleic acid immobilizing electrode 501 are retained at a prescribed reaction temperature (e.g., 20 to 40° C. for 30 to 60 minutes) so as to carry out the hybridization reaction. It is possible to dry the nucleic acid probe 502 for the preservation, and cleaned and sterilized gaseous materials such as nitrogen and the air are loaded in the detecting channel 531. In the stage of the hybridization reaction, the detecting channel 531 is loaded with a fluid containing the nucleic acid sample. In the nucleic acid detecting cassette of the closed system, it is impossible to discharge the gaseous material that is initially loaded to the outside. Therefore, the retreating channel 811d has an inner volume that permits retreating the gaseous material noted above. The inner volume of the retreating channel 811d in the initial state is substantially zero, and the retreating channel 811d is closed completely. Alternatively, it is possible to load a fluid such as a buffer solution or a physiological saline in the detecting channel 531 in place of the gaseous material.
(4)-2-5 Washing:
In the detecting channel 531, the nucleic acid immobilizing electrode 501 is washed after completion of the hybridization reaction, and the nucleic acid sample that was not involved in the hybridizing reaction is removed from the surface of the nucleic acid immobilizing electrode 501. In this process, a buffer solution is used as the washing solution. The washing solution used in this process is injected into the fluid holding channel 811e and is transferred into the detecting channel 531 when the washing solution is used. The total amount of about 48 μl of all the reagents are injected into the fluid holding channel 811e.
(4)-2-6 Electrochemical Measurement:
In the detecting channel 531, an intercalating agent (intercalator), which is a double stranded nucleic acid recognizing body that is selectively coupled with the hybridized the portion of double stranded nucleic acid is allowed to act on the hybridized nucleic acid sample after the washing stage so as to perform the electrochemical measurement. In this electrochemical measurement, a potential higher than the potential at which the intercalating agent carries out the electrochemical reaction is applied so as to measure the reaction current value derived from the intercalating agent. In this stage, the potential is swept at a constant rate. The detection of the target nucleic acid is judged on the basis of the current value thus obtained. Also, the temperature is maintained at, for example, 20 to 25° C. during the measuring process. The intercalating agent used is injected into the fluid holding channel 811f and is transferred into the detecting channel 531 when the intercalating agent is used. The total amount of about 48 μl of all the reagents are injected into the fluid holding channel 811f.
(4)-3 Fluid Transfer Order:
The nucleic acid detecting cassette 100 for the inspection is supplied to the user under the state that the required reagents are loaded therein as schematically shown in
(4)-3-1 Nucleic Acid Amplification:
In the first step, the sample containing nucleic acid is injected by opening the inlet-outlet vales 814 and 815. After injection of the sample, the inlet-outlet valves 814 and 815 are closed. Then, as shown in
(4)-3-2 Producing Single Stranded Nucleic Acid:
As shown in
(4)-3-3 Impartation of Protective Chain:
As shown in
(4)-3-4 Hybridization:
The hybridization process comprises (4a) hybridization, (4b) purging with air, part 1, (4c) transfer of the used reaction product, part 1, and (4d) transfer of the used reaction product, part 2.
(4)-3-4a Hybridization:
As shown in
In order to permit the nucleic acid probe 502 immobilized within the detecting channel 531 to be rendered sufficiently compatible with the nucleic acid sample transferred into the detecting channel 531 or in order to make the concentration of the nucleic acid sample uniform over the entire region of the detecting channel 531 in the stage of transferring the nucleic acid sample, it is possible to transfer the nucleic acid sample in the reciprocating fashion or in the pulse-wise transferring fashion in addition to the transfer of the nucleic acid sample at a constant fluid transfer velocity. In the case of employing the particular transfer method of the nucleic acid sample, it is possible to suppress the nonuniformity in the measured values of the current. In the reciprocating fashion of transfer the nucleic acid sample, the nucleic acid sample can be transferred from the detecting channel 531 toward the fluid holding channel 811c by pushing in the pushing pads of the retreating channel 811d, i.e., the central pad 401d, the left side pad 402d and the right side pad 403d. After completion of the transfer of the nucleic acid sample, the joining valves 833 and 835 are closed. Further, after the joining valves 833 and 835 are closed, the detecting channel 531 is maintained at a prescribed temperature so as to start up the hybridization reaction.
(4)-3-4b Purging with Air, Part 1:
As shown in
(4)-3-4c Transfer of Used Reaction Product, Part 1:
As shown in
(4)-3-4d Transfer of Used Reaction Product, Part 2:
As shown in
(4)-3-5 Washing:
The washing process includes (5a) washing and (5b) purging with air, part 2.
(4)-3-5a Washing:
As shown in
In order to remove without fail the nucleic acid sample that was not hybridized with the nucleic acid probe 502 from the surface of the nucleic acid immobilizing electrode 501 in the stage of transferring the washing solution, it is possible to transfer the washing solution in a reciprocating fashion or in a pulse-wise transfer fashion in addition to the transfer fashion at the constant fluid transfer velocity of the washing solution. In this case, it is possible to suppress the nonuniformity in the measured values of the current.
In the reciprocating fashion of transfer the washing solution, the washing solution can be transferred from the detecting channel 531 toward the fluid holding channel 811e by pushing in the pushing pad of the retreating channel 811d. After completion of the transfer of the washing solution, the joining valves 834 and 836 are closed.
(4)-3-5b Purging with Air, Part 2:
As shown in
(4)-3-6 Electrochemical Measurement:
The electrochemical measurement includes (6a) transfer of the intercalating agent, and (6b) transfer of the intercalating agent and electrochemical measurement.
(4)-3-6a Transfer of Intercalating Agent:
As shown in
(4)-3-6b Transfer of Intercalating Agent and Electrochemical Measurement:
As shown in
In order to allow the intercalating agent to act sufficiently on the hybridized nucleic acid probe 502 immobilized within the detecting channel 531, or in order to make uniform the concentration of the intercalating agent over the entire region of the detecting channel 531, it is possible to transfer the intercalating agent in a reciprocating fashion or in a pulse-wise transfer fashion in addition to the transfer fashion at a constant transfer rate. In this case, it is possible to suppress the nonuniformity in the measured values of the current.
In the reciprocating fashion of transfer the intercalating agent, the intercalating agent can be transferred from the detecting channel 531 toward the fluid holding channel 811e by pushing in the pushing pad of the retreating channel 811d. After completion of the transfer of the intercalating agent, the joining valves 834 and 836 are closed. After the joining valves 834 and 836 are closed, the detecting channel 531 is maintained at a prescribed temperature so as to start the electrochemical measurement.
(4)-3-7 Effect Produced by Consecutive Reaction Operation:
As described above, the a nucleic acid detecting closed cassette 100 having a variable-volume channel structure produces prominent effects as summarized below:
a. The reagent can be injected without causing a harmful air bubble to be involved in the reaction and in the transfer of the liquid material.
b. Since the joining channel can be arranged with the minimum length, the free space and the residual liquid material are scarcely held in the joining channel.
c. Since there is no pressure difference between the inside and the outside of the reagent fluid holding channel, the fluid leakage need not be worried about during storage of the reagent over a long period of time.
d. The fluid leakage need not be worried about during the transfer stage of the liquid material because there is no pressure difference in pressure between the inside and the outside of the reagent fluid holding channel.
e. The reagent and the reaction product can be mixed each other easily, and a satisfactory reaction can be carried out.
f. A complex pattern in the transfer of the liquid material can be employed in the hybridization process, the washing process and the reaction process with the intercalating agent so as to make it possible to suppress the final nonuniformity in the measured values of the current.
(5) Heat Transfer Unit:
The construction of the heat transfer unit will now be described in detail with reference to
(5)-1 Construction of Heat Transfer Unit:
As shown in
(5)-2 Contact of Heat Transfer Unit with Cassette:
(5)-3 Expansion of Flexible Sheet Toward Outside:
In order to moderate the pressure elevation inside the channel by the expansion of the flexible sheet 2 toward the outside of the nucleic acid detecting cassette 100 and in order to perform the heat transfer by maintaining the contact of the flexible sheet 2 with the contact pad 604a, it is necessary for the pushing spring 604a of the heat transfer block on the side of the flexible sheet 2 to be a spring of a low load. Preferably, a spring of a constant load is used as the pushing spring 604a noted above. It should also be noted that the requirements of the heat transfer to the flexible sheet 2 and the thermal expansion can be satisfied simultaneously as far as the contact pad 604a and the flexible sheet 2 are positioned close to each other even if the contact pad 604a and the flexible sheet 2 are not in mutual contact entirely or partially.
As described above, the flexibility of the flexible sheet 2 performs the three functions given below simultaneously:
a. The flexible sheet 2 is deformed toward the inner region of the channel so as to apply pressure to the fluid inside the channel. As a result, the fluid inside the channel is transferred to the adjacent fluid holding channel 111b via the joining channel 117.
b. The flexible sheet 2 is deformed toward the outside of the channel in accordance with the increase in volume of the fluid caused by, for example, the thermal expansion. As a result, the pressure elevation of the fluid inside the channel is moderated so as to prevent the fluid leakage.
c. The flexible sheet 2 is kept in good contact with or is positioned very close to the contact pad 604a even during the thermal expansion stage so as to carry out the heat transmission.
(5)-4 Cooling of Adjacent Fluid Holding Channel:
(5)-5 Series-Connected Channels:
Also, the reagent inside the fluid holding channel 811c, particularly, the nucleic acid probe inside the detecting channel 531, is weak against heat. Therefore, it is necessary to cool not only the adjacent fluid holding channel 811b but also the fluid holding channel 811c and the detecting channel 531 when the amplification of nucleic acid is being carried out. As shown in
(5)-6 Effect of Heat Transfer Unit:
Prominent effects can be produced by the combination of the heat transfer units 600, 610 with the nucleic acid detecting cassette 100 having a variable-volume channel structure as summarized in the following:
a. Since the channels are arranged in series, the temperature elevation of the unused reagent can be suppressed by simply cooling the adjacent fluid holding channel.
b. Since the pushing block used in a variable-volume channel is movable, it is possible to set the pushing block under the completely opened state. As a result, it is possible to achieve the thermal transfer from both sides of the fluid holding channel so as to suppress the heat loss.
(6) Entire Structure of Automatic Control Apparatus:
(6)-1 Automatically Controlling Constituent:
(6)-1-1 Block Diagram:
(6)-1-2 Motion Control:
The actuator driver 755 is formed of, for example, a stepping motor driver. The actuator driver 755 serves to drive a fluid transferring actuator 762 and a detachable actuator 763 based on the position of the nucleic acid detecting cassette 100 detected by a position sensor 764 so as to transfer the fluid or perform the attaching-detaching operation. The position sensor 764, which is not particularly shown in the drawings showing the construction of the system, is arranged in the vicinity of the moving positions of driving units 701 and 702, which are described herein later, of the nucleic acid detecting cassette 100. The fluid transferring actuator 762 and the detachable actuator 763 are realized by the driving units 701 and 702 in this embodiment of the present invention.
(6)-1-3 Temperature Control:
The temperature control driver 756 serves to control a heater/cooler 765 based on the temperature detected by the temperature sensor 766 so as to control the temperature. The heater/cooler 765 is realized by the heat transfer units 600 and 610 in this embodiment of the present invention.
(6)-1-4 Current Measuring Control:
The current measuring driver 757 takes out a current signal via an electric connector 703 connected to the nucleic acid detecting cassette 100 that is supported by a cassette holder 721 so as to gain the current signal via a current measuring terminal section 761.
(6)-2 Motion Control Mechanism:
Each of
(6)-2-1 X-Direction Motion Control:
As shown in
Arranged in the operating section are an electric connector 703, two driving units 701, 702, and the two heat transfer units 600, 610 as objects to be driven. The operating section can be moved freely in the X-, Y- and Z-directions by the driving system in the X-direction referred to above and by the driving systems in the Y- and Z-directions.
(6)-2-2 Y-Direction Motion Control:
The driving system in the Y-direction comprises a stationary Y-stage 731, a movable Y-stage 713a that can be moved in the Y-direction relative to the stationary Y-stage 731 by a Y-driving device 732 so as to have the position determined in the Y-direction, and a movable mounting plate 713b that is moved together with the movable Y-stage 713a. The stationary Y-stage 731 is fixed to the movable X-stage 723. It follows that the stationary Y-stage 731 can be moved in the X-direction together with the movement of the movable X-stage 723 in the X-direction.
(6)-2-3 Z-Direction Motion Control:
A first driving system in the Z-direction comprises a stationary Z-stage 725a and a movable Z-stage 726a that can be driven by a first Z-driving device 711 such that the position of the movable Z-stage 726a can be determined in the Z-direction relative to the stationary Z-stage 725a. The stationary Z-stage 725a is fixed to a first Z-stage mounting plate 724a that is fixed to a movable mounting plate 713b. As a result, the stationary Z-stage 725a can also be moved in the X- and Y-directions together with the movement of the first Z-stage mounting plate 724a in the X- and Y-directions.
A second driving system in the Z-direction comprises a stationary Z-stage 725b and a movable Z-stage 726b that can be driven by a second Z-driving device 712 such that the position of the movable Z-stage 726b can be determined in the Z-direction relative to the stationary Z-stage 725b. The stationary Z-stage 725b is fixed to a second Z-stage mounting plate 724b that is fixed to a movable mounting plate 713b. As a result, the stationary Z-stage 725b can also be moved in the X- and Y-directions in accordance with the movement of the second Z-stage mounting plate 724b in the X- and Y-directions.
(6)-2-4 Related Motion Control:
The driving units 701 and 702 are fixed to the movable Z-stages 726a and 726b via the mounting plates 727a and 727b, respectively. As a result, the driving units 701 and 702 can be moved freely in the X-, Y- and Z-directions in accordance with the movement of the movable Z-stages 726a and 726b in the X-, Y- and Z-directions.
Also, the heat transfer units 600 and 610 are fixed to the movable Z-stages 726a and 726b via the mounting tables 741 and 742 and, further, via the mounting plates 727a and 727b such that the heat transfer units 600 and 610 are selectively movable. Similarly, the electric connector 703 is also fixed to the movable Z-stages 726a or 726b such that the electric connector 703 is positioned in the vicinity of the region right above the driving units 701 and 702 and is selectively movable. As a result, the heat transfer units 600, 610 and the electric connector 703 can also be moved freely in the X-, Y- and Z-directions together with the movement of the movable Z-stages 726a and 726b in the X-, Y- and Z-directions. It should be noted, however, that the heat transfer units 600, 610 and the electric connector 703 can be moved selectively in the Z-direction so as to make it possible to bring individually the heat transfer units 600, 610 and the electric connector 703 to positions in the vicinity of the nucleic acid detecting cassette 100 or into contact with the nucleic acid detecting cassette 100.
Two driving units 701 and 702 are arranged in the present invention and these two driving units can be driven individually. As a result, it is possible to realize the fluid transfer operation by allowing one of these driving units to perform the operation of pressurizing the pad and the other driving unit so as to release the pressurization. For example, it is possible to circulate the reagent within the fluid holding channel by pushing the center of the channel by using the driving unit 701 and by releasing the pressurization on the left side of the fluid holding channel by using the driving unit 702.
Similarly, the two heat transfer units 600 and 610 are arranged in the present invention, and the temperature of these two heat transfer units can be individually controlled. As a result, it is possible to perform a unique control such that, for example, the heating is performed in one channel and the cooling is performed in the other channel.
(6)-3 Effect of Nucleic Acid Detecting System:
As described above, according to the nucleic acid detecting cassette 100 according to the first embodiment of the present invention and the nucleic acid detecting system equipped with the nucleic acid detecting cassette 100 as well as with the driving system and the control system for automatically controlling the nucleic acid detecting cassette 100, it is possible to automatically carry out continuously the series of operations including the amplification of nucleic acid and the other required processing and the detection of the target nucleic acid within a closed system.
(7) Modifications of First Embodiment:
Incidentally, the present invention is not limited to the first embodiment described above.
Specifically, the materials of the stationary base plate 1, the flexible sheet 2 and the cover plate 3 are not limited to those described previously.
Also, the present invention is not limited to the constructions of the blocks 101, 102, 103 and 106 shown in
The shape of the pad pushing each of the flexible sheets 2 is not limited to that in the first embodiment described above. In this embodiment, the pad of the fluid holding channel 111 or the retreating channel 131 consists of three pads. However, it is possible for the pad noted above to consist of at most two pads or at least four pads.
Also, in the first embodiment of the present invention, the opening-closing of each of the channels is controlled by using two driving units 701 and 702. However, it is also possible to use three or more driving units. Similarly, it is also possible to use many other heat transfer units in addition to the heat transfer units 600 and 610.
A second embodiment of the present invention, which corresponds to a modification of the first embodiment described with reference to
(1) Modification in Cassette Structure:
(1)-1 Channel Formed in Stationary Plate:
(1)-2 Channel Formed in Flexible Sheet:
(1)-3 Channel Formed in Each of Stationary Plate and Flexible Sheet:
(1)-4 Channel Formed by Expansion of Flexible Sheet
(1)-5 Coated Channel
Further,
Incidentally, the examples shown in
(2) Modifications of Channel Pattern:
(2)-1 One-Layer Structure Channel:
(2)-1-1 Structure of Each Channel:
As shown in
The fluid holding channel 791a is used as a reaction chamber for performing an amplification reaction of nucleic acid. The fluid holding channels 791a and 791b are joined to each other by a joining channel having the joining valve 792a mounted thereto. It is possible to inject a reagent and a sample into the fluid holding channel 791a via two valves 793a and 794a.
The fluid holding channel 791b is used as a reaction chamber for carrying out a reaction for the producing a single stranded nucleic acid. The fluid holding channels 791b and 791c are joined to each other by a joining channel having a joining valve 792b mounted thereto. It is possible to inject a reagent into the fluid holding channel 791b via a valve 794b.
The fluid holding channel 791c is used as a reaction chamber for carrying out a reaction for imparting a protective chain. The fluid holding channels 791c is joined to a detecting channel 791e by a joining channel having the joining valve 792c mounted thereto. It is possible to inject a reagent into the fluid holding channel 791c via a valve 794c.
The retreating channel 791d is used as a retreating channel of the detecting channel 791e. The retreating channel 791d is connected to the detecting channel 791d by a joining valve 792d. It is possible to inject a fluid into the retreating channel 791d via a valve 794d.
The detecting channel 791e is used as a reaction chamber for carrying out a reaction such as a hybridization reaction or for the detection. The fluid transferred in the detecting channel 791e is purged into the fluid holding channel 791g with gaseous material loaded in the retreating channel 791d. The fluid transferred in the detecting channel 791e is also purged into the fluid holding channel 791c with gaseous material loaded in the retreating channel 791f.
The retreating channel 791f is used as a retreating channel of the detecting channel 791e. The retreating channel 791f is joined to the detection channel 791e by a joining valve 792e. It is possible to inject a fluid into the retreating channel 791f via the valve 794f.
The fluid holding channel 791g is used as a chamber for holding a washing solution used for performing the washing treatment within the detecting channel 791e after the hybridization reaction. The fluid holding channel 791g is joined to the detecting channel 791e by a joining valve 792f. It is possible to inject a reagent into the fluid holding channel 791g via a valve 794g.
The fluid holding channel 791h is used as a chamber for holding a solution of an intercalating agent used for imparting the intercalating agent within the detecting channel 791e after the hybridization reaction and the washing treatment. The fluid holding channel 791h is joined to the detecting channel 791e by a joining channel having a joining valve 792g mounted thereto. It is possible to inject a reagent into the fluid holding channel 791h via a valve 794h or a valve 793b.
(2)-1-2 Effect of One-Layer Structure:
As shown in
Also, it is possible to modify the channel pattern in various fashions in addition to the construction shown in
Also, in the case of using a single flexible sheet 796 for forming each of the fluid holding channels 791a, etc. and the detecting channel 791e as in the example shown in
In the example according to the first embodiment of the present invention, the nucleic acid detecting chip 500 is immobilized on the glass base plate 500a. However, the present invention is not limited to the particular construction. For example, the nucleic acid detecting chip 500 can be made integral by forming the electrodes such as the nucleic acid immobilizing electrode 501, the counter electrode 503 and the reference electrode 504 on the stationary base plate 1. In the case of using the stationary base plate 795 shown in
It should also be noted that, in the example according to the first embodiment of the present invention, the nucleic acid detecting chip 500 is fixed to the stationary base plate 1. However, the present invention is not limited to the particular construction. In the case of using, for example, a glass base plate as the stationary base plate 1, it is possible to make the nucleic acid detecting chip 500 integral by forming the nucleic acid immobilizing electrode 501, the counter electrode 503 and the reference electrode 504 on the glass base plate.
(2)-2 Multiplex Detecting Apparatus:
Also, in the example according to the first embodiment of the present invention, the fluid holding channel and the retreating channel are arranged on a single line. However, it is also possible to arrange the fluid holding channels and the retreating channels on a plurality of straight lines so as to obtain a multiplex detecting apparatus.
(3) Effect and Modifications of Second Embodiment:
Further, as another channel pattern, the continuous channels can be formed by increasing or decreasing the number of reaction chambers. For example, in the construction exemplified in
As described above, according to the second embodiment of the present invention, which is achieved by modifying the construction according to the first embodiment of the present invention, it is possible to provide a nucleic acid detecting closed cassette that can be used for the automatic continuous processing throughout the system including the amplification of nucleic acid and other required processing and the detection of the target nucleic acid as in the first embodiment of the present invention.
The third embodiment corresponds to a modification in the shapes of the variable-volume channels such as the fluid holding channel and the retreating channel in each of the first and second embodiments described above. In the first embodiment, the channel is shaped substantially rectangular. However, it is possible to use, for example, a U-shaped variable-volume channel as in the third embodiment of the present invention. The construction substantially equal to that in the first embodiment is employed in the third embodiment unless otherwise specified.
(1) Basic Construction of Cassette:
As shown in
A plurality of U-shaped channels 913 whose inner volumes are variable are formed on the surface of the channel block 904. The adjacent U-shaped channels 913 are joined to each other by a joining valve 914. The fourth U-shaped channel 913 as viewed from the left side in the drawing performs the function of a retreating channel. The chip holder 901 that is made integral with the nucleic acid detecting chip 902 is fixed to the back surface of the fourth U-shaped channel 913 referred to above. Also, all of the U-shaped channels 913 are covered with the flexible sheet 905, and the seal block 906 is bonded to the flexible sheet 905. As a result, formed is the nucleic acid detecting cassette 900 shown in
(2) Channel System of U-Shaped Channel:
(2)-1 Entire Structure of Channel System:
(2)-2 Structure of the U-Shaped Channel:
(2)-3 Self-Sealing Type Port:
(2)-4 Seal Block:
(2)-5 Fluid Injection:
(2)-6 Individually Holding Section:
(3) Heat Transfer System:
After completion of the heating•cooling of the fluid holding channel 913a for amplifying nucleic acid in the process shown in
(4) Fluid Transfer System:
(4)-1 Fluid Transfer Module:
(4)-2 Fluid Transfer Process:
(4)-3 Residue Removing Filter:
(5) Effect of Third Embodiment:
As described above, according to the third embodiment of the present invention, which is achieved by modifying the construction according to the first and second embodiments of the present invention, it is possible to provide a nucleic acid detecting closed cassette that can be used for the automatic continuous processing throughout the system including the amplification of nucleic acid and other required processing and the detection of the target nucleic acid as in the first and second embodiments of the present invention.
According to the present invention, all the steps including the amplification of nucleic acid and other required processing and the detection of the target nucleic acid can be automatically carried out continuously without causing the air bubbles to be taken into the liquid material.
(Effect and Modifications of all Embodiments)
As described above, the present invention is effective in the technical field of a nucleic acid detecting closed cassette and a nucleic acid detecting apparatus that can be used for the automatic continuous processing throughout the system including the amplification of nucleic acid and other required processing and the detection of the target nucleic acid. The present invention is also effective in the technical field of a nucleic acid detecting system utilizing the particular nucleic acid detecting cassette and the nucleic acid detecting apparatus utilizing the particular nucleic acid detecting cassette.
Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.
Number | Date | Country | Kind |
---|---|---|---|
2003-400878 | Nov 2003 | JP | national |
Number | Name | Date | Kind |
---|---|---|---|
5229297 | Schnipelsky et al. | Jul 1993 | A |
5639428 | Cottingham | Jun 1997 | A |
5863801 | Southgate et al. | Jan 1999 | A |
6929239 | Colin et al. | Aug 2005 | B1 |
20030214057 | Huang | Nov 2003 | A1 |
Number | Date | Country |
---|---|---|
2 782 935 | Mar 2000 | FR |
8-62225 | Mar 1996 | JP |
2536945 | Jul 1996 | JP |
2573443 | Oct 1996 | JP |
9-511407 | Nov 1997 | JP |
WO 0053320 | Sep 2000 | WO |
WO 0132930 | May 2001 | WO |
Number | Date | Country | |
---|---|---|---|
20050153430 A1 | Jul 2005 | US |