JP4597091B2 - Biochemical analyzer and inspection cartridge used therefor - Google Patents

Description

  The present invention relates to a biochemical analyzer for analyzing a sample by flowing a test sample and a reagent in a test cartridge having a flow path and a reaction vessel formed on the surface thereof, and a test cartridge used therefor, and in particular, a test sample and a test cartridge using centrifugal force. It is suitable for those in which a reagent is flowed and mixed.

  Conventionally, in a biochemical analyzer that analyzes a specific biochemical substance from a sample containing a plurality of biochemical substances, it is known to flow a test sample and a reagent using centrifugal force. Are listed. Non-Patent Document 1 uses whole blood as a test sample, and after centrifuging whole blood in a test cartridge, the plasma component of whole blood and a reagent (diluent) are flowed into a mixing container and mixed together. . Mixing is performed by swinging the liquid in the circumferential direction in the mixing container by inertia force by rapidly decreasing the number of rotations of the rotating means during centrifugation. That is, when viewed in a moving coordinate system rotating on the same axis as the rotating means, the liquid in the container is shaken and mixed by greatly shaking the entire inspection cartridge.

JOURNAL OF AUTOMATIC CHEMISTRY, VOL. 17, no. 3, (1995), PP. 99-104, FIG.

  In the above prior art, in principle, since the liquid is swung by rocking the container, the liquid is swung as a whole and the mixing effect is small. Further, in order to cause the liquid to oscillate greatly, it is necessary to rapidly reduce or increase the number of revolutions to greatly oscillate the entire inspection cartridge and the rotating means, and a rotating means having a large rotational torque or braking torque is required. Become. Furthermore, centrifugal force is acting during mixing, and if the density difference between the liquids to be mixed is large, the liquid with a high density gathers on the outer periphery in the radial direction with respect to the rotating shaft in the container and mixes. Since the liquid to be separated is in a separated state, it becomes difficult to perform mixing.

The object of the present invention is to solve the above-mentioned problems of the prior art, enable efficient mixing of test samples or reagents regardless of the density difference between the liquids to be mixed, and mix and react many samples and reagents. , To perform detection automatically. Another object is to simplify and miniaturize the biochemical analyzer.
The present invention aims to achieve at least one of the above objects.

  The present invention that achieves the above object provides a biochemical analyzer for performing an analysis process by putting a sample and a reagent in a test cartridge having a flow path and a reaction container formed on the surface thereof and flowing them using centrifugal force. The test cartridge includes a mixing container that mixes the sample and the reagent, and a compressed air container that is connected to the mixing container and that compresses the internal gas by introducing the sample and the reagent in the mixing container by centrifugal force. An outer peripheral channel connected to the outer peripheral side of each of the mixing container and the compressed air container, and an inner peripheral channel connected to the outer peripheral channel from at least one of the mixing container and the compressed air container. It is a thing.

  Further, the present invention relates to a biochemical analyzer that performs an analysis process by putting a sample and a reagent in a test cartridge having a flow path and a reaction vessel formed on the surface thereof and using a centrifugal force to flow the test cartridge. A mixing container that mixes the sample and the reagent, a compressed air container that is connected to the mixing container and that compresses an internal gas by introducing the sample and the reagent in the mixing container by centrifugal force, and An outer peripheral flow path connected to the outer peripheral side of each of the mixing container and the compressed air container, and the outermost peripheral position of the compressed air container is arranged on the outer peripheral side of the outermost peripheral position of the mixing container It is.

  Furthermore, the present invention provides a test cartridge in which a flow path and a reaction container are formed on the surface, and a sample and a reagent are put into the test cartridge to perform analysis by flowing using a centrifugal force. A container, a compressed air container that is connected to the mixing container and in which the sample and the reagent in the mixing container are introduced by centrifugal force, and the gas inside is compressed, and the outer periphery of each of the mixing container and the compressed air container An outer peripheral channel connected to the side, and an inner peripheral channel connected to the outer peripheral channel from at least one of the mixing container and the compressed air container.

  Furthermore, the present invention provides a test cartridge in which a flow path and a reaction container are formed on the surface, and a sample and a reagent are put into the test cartridge to perform analysis by flowing using a centrifugal force. A container, a compressed air container that is connected to the mixing container and in which the sample and the reagent in the mixing container are introduced by centrifugal force, and the gas inside is compressed, and the outer periphery of each of the mixing container and the compressed air container An outer peripheral flow path connected to the side, and an outermost peripheral position of the compressed air container is disposed on an outer peripheral side of an outermost peripheral position of the mixing container.

  According to the present invention, the inner peripheral channel connected from at least one of the mixing container and the compressed air container is provided in the outer peripheral channel connected to the outer peripheral side of each of the mixing container and the compressed air container. Efficient mixing, and a large number of samples and reagents can be mixed, reacted, and detected.

Hereinafter, a biochemical analyzer and a cartridge used therefor according to an embodiment will be described with reference to the drawings.
FIG. 1 is a perspective view of a genetic analyzer 1 which is one of biochemical analyzers, and is a motor 11 arranged in a vertical axis, and a holder attached to an output shaft of the motor 11 and driven to rotate by the motor 11. And a disk 12. A large number of inspection cartridges 2 having the same shape are arranged in the circumferential direction of the holding disk 12. Above the inspection cartridge 2, a punching machine 13 is provided for punching a predetermined position of a flow path or a container formed in the inspection cartridge 2 and controlling the flow of the liquid. Further, a heating device 14 and a detection device 15 are disposed above the inspection cartridge 2.

In the analysis using the gene analyzer 1, the operator prepares the test cartridge 2 according to the test or analysis item and attaches it to the holding disk 12. In the mounted inspection cartridge 2, the reagent and the inspection sample flow through the flow path formed in the inspection cartridge 2 by the start and stop of the motor 11 and the operation of the punching machine 13. Then, genetic testing is performed.
2 and 3 show the details of the inspection cartridge 2. The inspection cartridge 2 has a fan-shaped portion with a conical tip and has a substantially trapezoidal shape. Is formed. An upper surface cover 199 (not shown) made of a film or a thin plate covers the entire upper surface of the test cartridge 2 in order to form a closed flow path or container for reagents or the like with the unevenness formed on the upper surface. The top cover 199 is bonded or bonded to the inspection cartridge 2. A plurality of reagent containers 230, 240, 270 to 290 that contain reagents necessary for the inspection are formed. As can be seen from FIG. 1, the upper side in FIG. 3 is the radially inner peripheral side with respect to the rotation axis of the holding disk 12. In the following, the inner peripheral side or the outer peripheral side indicates the radial positional relationship with respect to the rotation axis of the holding disk, and the upstream side or the downstream side indicates the inner peripheral side and the outer peripheral side, respectively.

  The reagent containers 230, 240, 270 to 290 are provided with outlet channels 231, 241, 271, 281 connected to the outer periphery thereof. In addition, since the reagent containers 280 and 290 have a common destination to which the reagent is moved, the outlet channels are shared. A sample container 310 is formed in the central portion of the fan-shaped portion to put a test sample into the test cartridge 2.

  Near the center of the test cartridge 2, there are a mixing container 420 for mixing and reacting the reagent and sample, a compressed air container 430 and an eluent container 270 arranged adjacent to the mixing container 420, and further on the outer peripheral side thereof. The coupling portion 401, the first amplification reagent container 280, and the second amplification reagent container 290 are further formed with an eluent recovery container 390 on the outer peripheral side thereof. On the outermost peripheral side, a waste liquid container 900 is formed over substantially the entire width of the inspection cartridge 2. Each container is connected by a flow path so that it can flow and react according to a predetermined procedure.

  The internal structure of the inspection cartridge 2 will be described with reference to FIG. The test cartridge 2 includes a container layer 198 in which a plurality of reagent containers and other containers are integrally formed, each of the plurality of reagent containers being sealed, and the flow of the reagent containers and other containers being controlled. The lid layer 190, the substrate layer 51 having a flow path for transferring the liquid after drilling, and the upper surface cover 199 covering the upper surface of the substrate layer 51 are laminated, and the layers are bonded or bonded together.

A predetermined amount of a necessary reagent is dispensed into each of the reagent containers 240 and 270 to 290 of the container layer 198 and sealed with an inner lid layer. In the inner lid layer 190, upper and lower connection holes 91 are provided where necessary, and the flow paths provided in the substrate layer 51 and the container layer 198 are connected via the upper and lower connection holes 91. In the inner lid layer 190, a plurality of perforated portions 89 are provided. The perforating unit 89 is sequentially perforated according to the processing procedure during the analysis process, and a predetermined flow operation is performed by sequentially connecting the flow paths or containers provided in the substrate layer 51 and the container layer 198.

A case where a necessary number of test cartridges 2 are mounted on the holding disk 12 and plasma is used as a sample to extract and analyze viral nucleic acid will be described with reference to the procedure manual shown in FIG. 5 and FIGS. 6 to 13.
(1) When starting the test, the operator centrifuges whole blood collected by a vacuum blood collection tube or the like into plasma and blood cell components, and injects the plasma into the sample container 310 from the sample inlet 301 of the test cartridge 2.
The reagent container 230, the cleaning liquid container 240, the elution liquid container 270, the first amplification liquid container 280, and the second amplification liquid container 290 are preliminarily provided with a solution, a cleaning liquid, an elution liquid, a first amplification liquid, a first Two amplification solutions are dispensed in a predetermined amount and are sealed by the inner lid layer 190 on the upper surface of the reagent container as described above.
(2) The punching machine 13 is used to punch two punching parts located at the upper part of the reagent container 230 (step 918 in FIG. 5; the same applies hereinafter).
The drilling operation will be described with reference to FIGS. FIG. 6 is an enlarged view of the vicinity of the reagent container 230, and FIGS. 7 and 8 show a cross section taken along the dotted line indicated by AA in FIG. The punching machine 13 is composed of a light source with high directivity and moves right above the punching parts 891 and 892, and then irradiates the punching parts 891 and 892 with light through the upper surface film (FIG. 7). The perforated portions 891 and 892 are made of a material having a high light absorption property, absorbs light when irradiated with light, becomes a high temperature, dissolves and is perforated. At this time, the top cover 199 is selected so as to have a high light transmittance and desirably a high temperature resistance, so that the top cover 199 is not perforated even when irradiated with light. In other words, the punching machine 13 can punch only the punching portions 891 and 892 existing inside the cartridge. In order to realize such perforation, a carbon dioxide laser or a high-power semiconductor laser is used as a light source, a thin resin film is used as the inner lid layer 190, and carbon is provided at predetermined positions on the inner lid layer 190 as the perforated portions 891 and 892. Can be applied or printed.

  When the holding disk 12 is rotated after the perforation (step 920), as shown in FIG. 8, the solution 238 flows toward the outer peripheral side by the action of centrifugal force, and exits from the perforated part 892 on the outer peripheral side. It flows into the mixing container 420 through the flow path 231. At this time, since the solution container outlet channel 231 is connected from the substrate layer 51 to the container layer 198 by the upper and lower connection portion 91, it flows into the mixing container 420 formed by the container layer 198 and the inner lid layer 190. Is possible. When the liquid flows out from the outer perforated portion 892, the inner perforated portion 891 allows air to flow into the reagent container 230 via the air channel 85 connected to other containers. As a result, the liquid flows out smoothly and can be replaced with air.

In the rotation of step 920, the plasma dispensed into the sample container 310 also flows into the mixing container 420 at the same time via the sample container outlet 311.
(3) In the mixing container 420, the mixed plasma and the solution are mixed and reacted (step 922).
The lysate dissolves the cell membrane from viruses, bacteria, etc. in plasma to elute the nucleic acid, and further promotes adsorption of the nucleic acid to the binding portion 401. Guanidine hydrochloride is used as a reagent for elution and adsorption of DNA, and guanidine thiocyanate is used as a reagent for elution and adsorption of RNA.
In order to dissolve cell membranes such as viruses and bacteria in plasma, it is necessary to sufficiently mix the lysate and plasma. The mixing is composed of a mixing container 420, a compressed air container 430, and flow paths 431 to 434. This is performed by utilizing the pressure generated by the connecting flow path and centrifugal force.

The operation will be described with reference to FIGS.
FIG. 9 is an enlarged view around the mixing container 420 and the compressed air container 430. Between the mixing container 420 and the compressed air container 430, the mixing container outer peripheral flow path 431 connected to the outer peripheral side of the mixing container 420, the mixing container inner peripheral flow path 432 connected to the right side of the mixing container 420, the compressed air container 430 The compressed air container outer peripheral flow path 433 connected to the outer peripheral side, the compressed air container inner peripheral flow path 434 connected to the left side of the compressed air container 430, and the connecting flow path constituted by the mixing means 436 are connected.
The mixing container outer peripheral flow path 431 and the mixing container inner peripheral flow path 432 merge at a merging branch point 437 and are connected to the mixing means 436. The compressed air container outer peripheral flow path 433 and the compressed air container inner peripheral flow path 434 merge at the merge branch point 438 and are connected to the mixing means 436. Therefore, these flow paths are all connected. Further, in the connection flow path, at least a part of all the paths connecting the mixing container 420 and the compressed air container 430 is located on the outer peripheral side from the compressed air container 430.

When sufficient centrifugal force is received by the rotation of the holding disk and the plasma and the lysate flow into the mixing container 420, the liquid moves so as to stick to the outer peripheral side of the mixing container 420, and the rotation axis of the holding disk is the center. The liquid surface 601 is formed in an arc shape. The connection channel is connected to the channel sealing portion 421 on the outer peripheral side. As shown in FIG. 3, the flow path sealing portion 421 connects the connection flow path on the container layer 198 and the flow path on the substrate layer 51 provided on the outer peripheral side thereof. The connection is sealed, and the liquid cannot flow from the flow path sealing portion 421 to the outer peripheral side. Therefore, plasma and lysate are held in the mixing container 420.

  Plasma has almost the same density as water because most of its components are water, whereas lysate is a reagent in which a large amount of salt is dissolved, so its specific gravity is heavier than plasma. . Therefore, as shown in FIG. 9, in the state where the centrifugal force is applied, the plasma and the lysis solution are separated into the plasma 308 and the lysis solution 238 in the mixing container 420. Since plasma and lysate flow at the same time, they are mixed somewhat in the process of flowing into the mixing container, and a layer composed of a mixture of plasma and lysate is formed between them. Therefore, it is in a separated state.

  Part of the plasma 308 and the lysate 238 flows into the compressed air container 430 via the connection flow path, and forms an arc-shaped liquid surface 603. Since a part of the path of the connection channel is formed outside the outermost peripheral portion of the compressed air container 430, once the liquid enters the connection channel, the gas (in this case, air) pre-existing in the compressed air container 430 Cannot exit the compressed air container 430. In other words, as shown in FIG. 9, when the liquid enters the compressed air container 430, the gas is compressed by a volume corresponding to the amount of liquid that has entered, forming a compressed gas 508.

In FIG. 9, the liquid level 601 in the mixing container 420 is on the inner peripheral side from the liquid level 603 in the compressed air container 430, and when the liquid is subjected to centrifugal force, the liquid level is equal to the compressed air container 430 side. When the liquid moves to the compressed air container side, the compressed gas 508 is further compressed, and a pressure for pushing back the liquid acts. The positions of the liquid level 601 and the liquid level 603 are determined so that these two actions are balanced, and the liquid level 603 is always located on the outer peripheral side from the liquid level 601.
Since the force for moving the liquid toward the compressed air container is generated by centrifugal force, this force increases when the number of rotations of the holding disk is increased. Therefore, when the rotation speed of the holding disk is increased from the state shown in FIG. 9, the liquid further moves from the mixing container 420 to the compressed air container 430, the liquid level 601 moves to the outer peripheral side, and the liquid level 603 moves to the inner peripheral side. To do. On the other hand, when the rotational speed is lowered, the liquid level 601 and the liquid level 603 move in opposite directions. 9 can be moved between the mixing container 420 and the compressed air container 430 by increasing or decreasing the rotational speed of the holding disk in the state shown in FIG.

The operation of the liquid when the rotational speed of the holding disk is increased from the state of FIG. 9 will be described with reference to FIGS.
Since the liquid moves from the mixing container 420 toward the compressed air container 430, as shown in FIG. 10, the mixing container inner peripheral flow path 432 is connected to the mixing container 420 on the inner peripheral side where plasma exists. The plasma flows in the direction indicated by the arrow in the figure, the dissolution liquid flows in the mixing container outer peripheral flow path 431, the two liquids merge at the merge branch point 437, and flow into the mixing means 436. . Although both are mixed by merging, mixing can be further promoted by providing the mixing means 436. If the mixing means 436 embeds, for example, a porous resin on the flow path, the mixing is promoted by causing the liquid to flow in a complicated manner between minute voids. As another example, one or a plurality of protrusions may be provided so that the flow collides with the flow path.

  Even if the mixing means 436 is not provided, the plasma and the lysate are combined and flowed, so that a certain amount of mixing can be performed due to the turbulent flow at the merging portion or the diffusion phenomenon on the merged flow path. Further, the mixed liquid flows into the compressed air container via the compressed air container outer peripheral flow path 433. Since the density of the liquid mixed at this time is intermediate between the lysate and plasma, it flows into the lysate layer in the compressed air container 430 and then moves to the water level 604 at the boundary between the plasma and lysate. In this process, liquid is further mixed.

  Further, in the present embodiment, the channel cross-sectional area of the mixing container outer peripheral channel 431 is smaller than the channel cross-sectional area of the mixing container inner peripheral channel 432. Thereby, the flow channel resistance of the mixing container outer peripheral flow channel 431 is larger than that of the mixing container inner peripheral flow channel 432. The difference in flow path resistance is set in consideration of the density difference between plasma and lysate. The centrifugal force acting on the liquid per unit volume is ρ, the distance from the center of rotation is r, and the rotational angular velocity is ω. Since centrifugal force = ρrω ^ 2, the centrifugal force acting when the liquid density is heavy is Become stronger.

In FIG. 10, when the path L1 on the mixing container inner flow path 432 and the path L2 in the mixing container 420 are compared, the relationship in the radial position from the rotation center of the holding disk is the same, but the types of liquids are different. The centrifugal force acting on the liquid (plasma) in the container inner circumferential channel 432 is slightly reduced in proportion to the density. For this reason, by increasing the flow path resistance of the mixing container outer peripheral flow path 431, it is possible to make the solution 238 difficult to flow and to set both to flow at an appropriate ratio. That is, the difference in density is canceled by the channel resistance, and liquids having different densities can be joined at a predetermined ratio.
If there is a difference in viscosity between plasma and lysate, and the viscosity of the lysate is relatively high, the lysate will not flow easily without changing the channel resistance, but the same effect can be obtained. Samples vary greatly in viscosity from sample to sample, and conditions where the viscosity of the solution is always relatively high cannot be expected. Even in such a case, by providing a difference in flow path resistance, a large amount of plasma on the inner peripheral side can be guided to the junction. Even when there is no difference in channel resistance, the amount of plasma flowing is relatively small, and the mixing effect can be obtained although the effect is inferior.

When the number of rotations of the holding disk is increased, the mixing action by the plasma and the lysate flowing from the mixing container inner peripheral flow path 432 flows from the mixing container inner peripheral flow path 432 and the mixing container outer peripheral flow path 431 and merges at the junction branch point 437 will be described below. Set to 1.
When the number of rotations of the holding disk is further increased, a state as shown in FIG. 11 is obtained. The liquid level 601 in the mixing container further moves to the outer peripheral side, and decreases from the mixing container inner peripheral flow path 432 to the outer peripheral side. In this state, since the liquid cannot flow into the mixing container inner peripheral flow path 432, only the solution 238 moves from the mixing container outer peripheral flow path 431. As the liquid flows in, the water level on the compressed air container 430 side moves to the inner peripheral side, and the dissolved liquid flows into the compressed air container 430 from both the compressed air container outer peripheral channel 433 and the compressed air container inner peripheral channel 434. At this time, similarly to the mixing action 1, the compressed air container outer peripheral channel 433 has a channel resistance larger than that of the compressed air container inner peripheral channel 434, so that the dissolved solution overcomes the density difference and flows from the compressed air container inner peripheral channel 434. To do. At this time, the dissolved solution flows into the layer of plasma 308 in the compressed air container 430 so that the dissolved solution and the plasma are mixed. This is referred to as mixing action 2.

The operation of the liquid when the rotational speed of the holding disk is lowered from the state of FIG. 11 will be described with reference to FIGS.
When the rotation speed is lowered, the liquid moves from the compressed air container 430 to the mixing container 420. The operation is obtained by replacing the relationship between the mixing container 420 and the compressed air container 430 in the description of the mixing action 1. That is, the solution flows from the compressed air container outer peripheral flow path 433, and the plasma flows from the compressed air container inner peripheral flow path 434, and both merge at the merge branch point 438 and are mixed by the mixing means 436. The mixed liquid flows into the mixing container 420 from the mixing container outer peripheral channel 431 and further mixes with the solution in the mixing container 420.

  Similar to the mixing action 1, the compressed air container outer peripheral flow path 433 has a larger flow resistance than the compressed air container inner peripheral flow path, and thereby moves the plasma in the compressed air container 430 against the density difference. be able to. This is referred to as mixing action 3.

  When the rotational speed is further lowered from the state of FIG. 12, only the solution 238 moves from the compressed air container 430 to the mixing container 420 via the compressed air container outer peripheral channel 433 as shown in FIG. 13. Similar to the mixing action 2, the lysing solution 238 can flow into the mixing container 420 from the mixing container inner peripheral flow path 432 and is mixed with the plasma in the mixing container. This is referred to as mixing action 4.

If the series of operations shown in FIGS. 10 to 13 is a unit operation, not all liquids are mixed in one unit operation, but all liquids can be mixed by repeating the unit operation. That is, the partially mixed liquid has an intermediate density between the plasma and the lysate, and moves in the vicinity of the intermediate water levels 602 and 604 between the plasma and the lysate in the mixing container or the air compression container. Next, when the unit operation is performed, liquid that is not relatively mixed is present at the position of the connection flow path, and thus the liquid that is not mixed is preferentially mixed.
Although only a part of the liquid is mixed in one unit operation, all the liquids are mixed by repeating the unit operation. Accordingly, the amount of liquid that moves in one unit operation can be reduced, and the entire mixing can be realized with a small compressed air container.
In particular, the configuration is such that the connection position of the mixing container inner peripheral flow path 432 is on the inner peripheral side from the liquid level 601 in the mixing container when the rotational speed is increased (see FIG. 11), so that the water level 601 is set on the outer peripheral side. When moving, the most unmixed (low density) plasma in the vicinity of the liquid surface 601 is surely flowed and mixed in the mixing container inner circumferential flow path 432. Similarly, the liquid level 603 is configured such that the connection position of the compressed air container inner peripheral flow path 434 is located on the inner peripheral side of the compressed air container inner liquid level 603 when the rotational speed is lowered (see FIG. 13). Nearly unmixed (smallest density) plasma in the vicinity flows into the compressed air container inner circumferential flow path 434 and receives the mixing action.

  In addition, when there is no density difference between the two liquids to be mixed, the two liquids can freely flow in the container even under a centrifugal force, so when the liquid flows into the mixing container or the compressed air container from the connection flow path. It is easily mixed by the induced flow.

  The speed at which the rotational speed is increased or decreased may be approximately the same as the speed at which the liquid moves, and a rapid change is not required as in the case where the inspection cartridge is swung and mixed. For this reason, a rotating means having a low rotational torque or braking torque can be used. On the other hand, the speed at which the liquid moves can be moderated by making the connecting flow path thinner overall, and can be adjusted according to the capacity of the motor.

  In addition, in the mixing operation, it is not necessary for both containers to have connection flow paths at two water levels, and if the connection flow path is connected to either container at two water levels, mixing is possible. An effect can be obtained. That is, even if there is no compressed air container inner peripheral flow path 434, the mixing action 1 and the mixing action 4 work, so that a mixing effect can be obtained. On the other hand, since the mixing action 2 and the mixing action 3 act even in the absence of the mixing container inner circumferential flow path 432, a mixing effect can be obtained.

  Furthermore, although the connection flow path is provided at two water levels for both containers, it may be provided at three or more water levels. When there is almost no difference in density between the liquids to be mixed, the flow can be induced in a wider range when the liquid flows into the mixing container or compressed air container by connecting at many water levels. Mixing is promoted. When the liquids to be mixed are three types of liquids having different densities, it is possible to efficiently mix by providing three connection flow paths at positions corresponding to the three types of liquids.

  In addition, all of the liquids to be mixed are liquids, but the solid reagent and the liquid may be mixed by arranging a solid reagent in the mixing container, the compressed air container, or the connection channel in advance. Is possible. In this case, when the liquid flows into the mixing container, the solid reagent comes into contact with the liquid and starts to dissolve in the surrounding liquid. And the density of the liquid of the part which the solid reagent contacted rises, As a result, a density difference generate | occur | produces in a liquid. The liquid having the density difference travels between the mixing container and the air compression container, and is mixed and uniformed. For this reason, dissolution of the solid reagent is promoted. In particular, when a solid reagent is disposed on the connection channel, dissolution is further promoted by the flow in the channel.

After repeating the unit operation as many times as necessary for mixing, when the reaction between the plasma and the lysate is completed, the rotation of the holding disk 12 is stopped (step 924). Hereinafter, a solution in which plasma and a lysis solution are mixed and the reaction is completed is referred to as a lysis mixture.
(7) Transition to combined mode.
The perforation part 89 on the inner lid layer 190 of the flow path sealing part 421 is perforated by the perforator 13 (step 926). The holding disk 12 is rotated (step 928). Since the mixing vessel on the container layer 198 is connected to the channel on the substrate layer 51 by perforating the channel sealing unit 421, the dissolved mixed solution flows into the coupling unit 401 provided on the outer peripheral side. To do.
As shown in FIG. 3, the binding portion 401 is formed at a substantially central portion of the test cartridge 2 and is filled with a binding filter for binding nucleic acids. When the dissolved mixed liquid that is the mixed liquid passes through the binding unit 401 (step 930), the nucleic acid is adsorbed to the binding filter provided in the binding unit 401. The waste liquid generated after passing through the coupling portion 401 is further directed to the outer peripheral side. However, since the flow path sealing portion 402 is provided, the liquid is transferred to the waste liquid port 900. The configuration of the channel sealing unit 402 is the same as that of the channel sealing unit 421 described above. The rotation of the holding disk is stopped (step 932).
(8) Transition to the cleaning mode.
In the same procedure as the solution, the two perforations 89 on the cleaning liquid container 240 are perforated (step 934). The holding disk 12 is rotated again (step 936), and the cleaning liquid 240 in the cleaning liquid container is guided to the mixing container 420 and the connecting portion 401 by centrifugal force (step 938). The mixing container 420 is cleaned and the coupling portion 401 is cleaned. The cleaning liquid is transferred to the waste liquid port 900 in the same manner as the solution.
As the cleaning liquid, for example, ethanol or an aqueous ethanol solution is used to clean unnecessary components such as salt attached to the compressed air container 430 and the coupling portion 401. Further, if necessary, a plurality of cleaning liquids may be used for repeated cleaning.

Next, in order to shift to the nucleic acid elution step, the holding disk 12 is stopped (step 940).
(9) Transition to the elution mode.
The punching part 89 on the inner lid layer 190 of the flow path sealing part 402 is punched by the punching machine 13 (step 941). By piercing the flow path sealing portion 402, the coupling portion 401 on the container layer 198 is connected to the flow path on the substrate layer 51, and the eluent recovery container 390 provided on the outer peripheral side of the coupling portion 401 Connected.
Two perforations 89 on the eluent container 270 are perforated by the same procedure as that for the lysing solution (step 942). The holding disk 12 is rotated (step 944), and the eluent is caused to flow through the coupling portion 401.
As the eluent, water or an aqueous solution whose pH is adjusted to 7 to 9 is used. The eluent elutes the nucleic acid from the binding filter of the binding portion 401 (step 946). As shown in FIG. 3, the flow path 411 directed toward the waste liquid port 900 is once configured to be directed toward the inner peripheral side, so that the eluent containing nucleic acid is transferred to the eluent recovery container 390 instead of the waste liquid port. And retained. The rotation of the holding disk 12 is stopped (step 948).
(10) Transition to amplification and detection mode.
Two perforations 89 located at the top of the first amplification liquid container 280 are perforated by the same procedure as that for the lysate. When the holding disk 12 is rotated, the first amplification liquid flows into the eluent recovery container 390. The first amplification solution is a reagent that amplifies and detects a nucleic acid, and includes, for example, deoxynucleoside triphosphate and a fluorescent reagent. Similar to the eluent, the first amplification liquid is held in the eluent recovery container 390. The holding disk 12 is stopped.
After circulating the first amplification solution, the heating device 14 is moved to the eluent collection container of the inspection cartridge. Alternatively, the holding disk 12 is rotated and the inspection cartridge 2 is moved to the position of the heating device 14. The temperature of the eluent recovery container 390 is controlled using the heating device 14.
Next, two perforations 89 located at the top of the second amplification solution container are perforated by the same procedure as that for the lysis solution. The holding disk 12 is rotated. Due to the centrifugal force, the second amplification liquid flows into the eluent recovery container 390. When the second amplification liquid is transferred to the eluent recovery container 390, the heating device 14 is moved to the eluent recovery container 390 of the inspection cartridge or the holding disk 12 is rotated to bring the inspection cartridge 2 to the position of the heating apparatus 14. Move. The temperature of the eluent recovery container 390 is controlled. While the temperature is controlled for a predetermined time, the nucleic acid is amplified (step 950), and the detection device 15 detects the nucleic acid (step 952). Heating is performed for a time required for amplification and detection, for example, about 30 minutes to 2 hours, and the test is terminated (step 954).

  The disk is temporarily stopped at the time of drilling. However, it is not always necessary to stop the disk. For example, when drilling is performed with a laser having a short irradiation time, the predetermined position may be drilled in synchronization with the rotation. Is shortened. Even in the configuration in which the plasma and the lysate are mixed, it is not necessary to stop the holding disk because the mixing is possible only by raising and lowering the centrifugal force. For this reason, the liquid does not move to an undesired place due to capillary action at the time of stoppage, and the analysis operation is not hindered.

As described above, the connecting flow path includes the mixing container and the compressed air container, and is connected to at least one of the mixing container and the compressed air container at at least two positions in the radial direction when viewed from the rotating shaft of the rotating means. By providing this, mixing can be performed with a simple inspection cartridge by at least one of the mixing operations 1 to 4. Moreover, even if there is a density difference between the liquids to be mixed, mixing can be performed.
Further, since it is not necessary to perform rapid acceleration / deceleration as in the case where the inspection cartridge is swung and mixed, a rotating means having a low rotational torque or braking torque can be used, and thus the analyzer can be simplified.
Furthermore, since all liquids are mixed by repeating the unit operation even though only a part of the liquid moves in one unit operation, the compressed air container can be made small, and a small inspection cartridge can be used. Mixing can be realized. In general, the rotational torque required to rotate the disk is proportional to the fifth power of the outer diameter in consideration of the air resistance of the disk during rotation. Therefore, since mixing can be realized with a small test cartridge, the required rotational torque is also reduced, and the motor mounted on the analyzer can be miniaturized. As a result, a small analyzer can be realized.
The connection position of the flow path connected on the innermost peripheral side is the position of the liquid surface formed in the source container when the liquid in the mixing container or the compressed air container moves to the other container due to a change in centrifugal force. Further, by being located on the inner peripheral side (see FIGS. 11 and 13), the least mixed (low density) liquid in the vicinity of the liquid surface position can be surely guided to the connection flow path to give a mixing action. Therefore, all the liquids can be mixed more reliably by repeating the unit operation.

Furthermore, the flow path resistance from the connection position of the flow path connected at the outermost peripheral side to the merge branch point is larger than the flow path resistance from the connection position of the flow path connected at other positions to the merge branch point. Therefore, the difference in the density of the liquids to be mixed can be canceled by the channel resistance, and the liquids having different densities can be joined at a desired ratio. Furthermore, if a mixing means is provided on the flow path merged from the merge branch point or the merge branch point, the mixing can be performed more efficiently.
FIG. 14 shows an inspection cartridge 2 according to another embodiment.
FIG. 15 is an enlarged view of the vicinity of the mixing container 420 and the compressed air container 430. Between the mixing container 420 and the compressed air container 430, a connection channel 439 that connects the outermost peripheral side of the mixing container 420 and the outermost peripheral side of the compressed air container 430 is provided. Further, the outermost peripheral position of the compressed air container 430 is arranged on the outer peripheral side of the outermost peripheral position of the mixing container 420.
When the plasma and lysate flow into the mixing container 420 due to the centrifugal force due to the rotation of the holding disk, the liquid moves so as to stick to the outer peripheral side of the mixing container 420 and has an arc shape centering on the rotation axis of the holding disk. A liquid surface 601 is formed on the surface. Part of the plasma and the lysate flows into the compressed air container 430 via the connection flow path, and becomes an arc-shaped liquid surface 603 centered on the rotation axis of the holding disk.

When the number of rotations of the holding disk is increased, the liquid moves from the mixing container 420 toward the compressed air container 430 as shown in FIG. 16 from the state of FIG. Since the connection channel 439 is connected on the outer peripheral side of the mixing container 420, only the solution flows into the compressed air container 430. However, since the solution is still present on the outer peripheral side of the compressed air container 430, the plasma and the solution are not mixed at this time.
When the number of rotations of the holding disk is further increased, a state as shown in FIG. 17 is obtained. When all the lysate held on the outer peripheral side in the mixing container 420 moves to the compressed air container 430, only the plasma 308 exists in the mixing container 420, so that the plasma 308 passes through the connection flow path 439 and is compressed air. Move to container side.

Since the dissolved solution that has flowed in first exists on the outer peripheral side of the compressed air container 430, the plasma that has flowed into the compressed air container from the connection flow channel flows into the dissolved solution layer and mixes with the dissolved solution.
When the rotation number of the holding disk is further increased, almost all plasma can be moved to the compressed air container side as shown in FIG.
The compressed air container is arranged on the outer peripheral side from the mixing container in the radial direction from the rotating shaft of the holding disk. As a result, as shown in FIG. 18, since the liquid level 601 can be maintained on the inner peripheral side from the liquid level 603 even after a large amount of liquid has moved, all liquids are moved to the compressed air container side by centrifugal force. As a result, all plasma and lysate can be mixed.

  When the number of rotations of the holding disk is lowered, a reverse flow from the compressed air container 430 to the mixing container 420 occurs, and the plasma and the lysate are mixed. By lowering or stopping the rotation speed of the holding disk as much as possible, all the liquid can be moved to the mixing container side, and all plasma and lysate can be removed in the same way as when the rotation speed is increased. Can be mixed again. Since the compressed air container is arranged on the outer peripheral side from the mixing container, the inspection cartridge is slightly larger than the first embodiment, but mixing can be performed with a simpler configuration.

In addition, when a series of operations for increasing and decreasing the number of revolutions is a unit operation, all plasma is mixed by flowing into the lysate at least once, but the mixing is insufficient in a single unit operation. In this case, it is preferable to repeat the unit operation as many times as necessary.
Further, even when the speed of increasing and decreasing the rotational speed is very slow, the order in which the plasma and lysate flow does not change at all, so a rapid change is not required as in the case of mixing by shaking the test cartridge. For this reason, a rotating means having a low rotational torque or braking torque can be used. Furthermore, the solid reagent can be dissolved and mixed by arranging the solid reagent in advance on a mixing container, a compressed air container, or a connection channel.
As described above, mixing, reaction, and detection of a large number of samples and reagents can be automatically executed by a simple and small inspection cartridge and a biochemical analyzer.

The perspective view which shows the gene diagnostic apparatus which concerns on one embodiment by this invention. The perspective view of the inspection cartridge concerning one embodiment (state which removed the upper surface cover). The top view of the test | inspection cartridge which concerns on one Embodiment. The disassembled perspective view which shows the assembly of the test | inspection cartridge which concerns on one embodiment. The flowchart which shows the operation | movement procedure of the genetic diagnostic apparatus which concerns on one embodiment. The partial top view of the test | inspection cartridge which concerns on one embodiment. The fragmentary sectional view explaining the perforation of the reagent container concerning one embodiment (at the time of perforation). The fragmentary sectional view explaining the drilling of the reagent container which concerns on one embodiment (after drilling). The partial top view of the test | inspection cartridge which concerns on one embodiment. The partial top view of the test | inspection cartridge which concerns on one embodiment. The partial top view of the test | inspection cartridge which concerns on one embodiment. The partial top view of the test | inspection cartridge which concerns on one embodiment. The partial top view of the test | inspection cartridge which concerns on one embodiment. The top view which shows the test | inspection cartridge concerning other embodiment. The fragmentary top view of the test | inspection cartridge which concerns on other embodiment. The fragmentary top view of the test | inspection cartridge which concerns on other embodiment. The fragmentary top view of the test | inspection cartridge which concerns on other embodiment. The fragmentary top view of the test | inspection cartridge which concerns on other embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Gene analyzer, 2 ... Test cartridge, 11 ... Motor, 12 ... Holding disk,
DESCRIPTION OF SYMBOLS 13 ... Punching machine, 14 ... Heating apparatus, 15 ... Detection apparatus, 90 ... Lid, 199 ... Top cover, 230 ... Reagent container, 420 ... Mixing container, 430 ... Compressed air container, 431 ... Mixing container outer periphery flow path,
432 ... mixing container inner peripheral flow path, 433 ... compressed air container outer peripheral flow path, 434 ... compressed air container inner peripheral flow path, 436 ... mixing means.

Claims (5)

In a biochemical analyzer that performs analysis processing by putting a sample and a reagent in a test cartridge formed on the surface with a flow path and a reaction container and using centrifugal force to flow,
The test cartridge includes a mixing container that mixes the sample and the reagent, and compressed air that is connected to the mixing container and in which the sample and the reagent in the mixing container are introduced by centrifugal force to compress an internal gas. A container,
An outer peripheral flow path connected to the outer peripheral side of each of the mixing container and the compressed air container;
An inner circumferential channel connected to the outer circumferential channel,
One end of the inner peripheral flow path is connected to at least one of the mixing container and the compressed air container on the inner peripheral side from the connection position of the outer peripheral flow path, and the other end of the inner peripheral flow path is connected to the outer peripheral flow path. biochemical analysis apparatus characterized by there. The biochemical analyzer according to claim 1,
One end of the inner peripheral container is connected to the mixing container,
Furthermore, a biochemical analyzer comprising a mixing means on the compressed air container side from a junction branch point between the inner peripheral container and the outer peripheral flow path. The biochemical analyzer according to claim 1,
One end of the inner peripheral container is connected to the mixing container,
The biochemical analyzer according to claim 1, wherein a flow path resistance from the mixing container to the junction branch point with the inner peripheral container in the outer peripheral flow path is made larger than a flow path resistance of the inner peripheral flow path. The biochemical analyzer according to claim 1,
A biochemical analyzer characterized in that a solid reagent is disposed in advance in at least one of the mixing container, the compressed air container, the outer peripheral channel, and the inner peripheral channel. In a test cartridge that is formed on the surface with a flow path and a reaction container, and performs analysis processing by flowing a sample and a reagent using centrifugal force,
A mixing container for mixing the sample and the reagent;
A compressed air container that is connected to the mixing container and in which an internal gas is compressed by introducing the sample and the reagent in the mixing container by centrifugal force;
An outer peripheral flow path connected to the outer peripheral side of each of the mixing container and the compressed air container;
An inner circumferential channel connected to the outer circumferential channel,
One end of the inner peripheral flow path is connected to at least one of the mixing container and the compressed air container on the inner peripheral side from the connection position of the outer peripheral flow path, and the other end of the inner peripheral flow path is connected to the outer peripheral flow path. Inspection cartridge characterized by.

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