JP2008268198A - Separation chip and separation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a separation means for efficiently separating insoluble components, such as blood corpuscles, using a small-sized device. <P>SOLUTION: A separation chip is constituted so as to be successively rotated at a first rotational speed and a second rotational speed higher than the first rotational speed, with respect to the rotary shaft outside the chip to separate insoluble components from a suspension by centrifugal force and gravity and has a first liquid storage tank which stores the introduced suspension and holds the insoluble component separated from the suspension by centrifugal force and gravity and a separation flow channel for allowing components, other than the insoluble component in the suspension separated by centrifugal force and gravity, to flow out. The separation flow channel is connected to the first liquid storage tank and stretched upward from the connection position toward the inner periphery, from a direction vertical to the resultant of the centrifugal force and gravity at the first rotational speed. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a separation chip and a separation method.

  Conventionally, most of the analysis of trace molecules related to clinical diagnosis, food hygiene, and environmental analysis is performed using a mass spectrometer after separating a sample using a centrifuge, gas chromatography, liquid chromatography, etc. Analysis is being performed. Since these analyzers are expensive and require specialized knowledge to operate, analysis has been performed by clinical laboratory companies and analysis companies. In recent years, as a trend of the world, simple and quick diagnosis at the bedside, analysis and measurement at each site of food processing and import, to prevent accidents and analyze harmful substances in rivers and waste The importance of performing on-site such as rivers and waste disposal sites has attracted attention, and the development of detection methods and analyzers that can be measured easily, quickly, inexpensively and with high sensitivity is emphasized.

  In particular, in the analysis for clinical diagnosis, the analysis time is shortened and the amount of the sample required for the analysis is reduced, and at the same time, in order to detect the disease state at an early stage, it is detected with a high sensitivity using a small amount of sample. This is an important issue. However, in the current general analysis method, the test sample is separated using the above-mentioned apparatus, and then the sample components including the measured substance are extracted and analyzed manually or using a dispenser. There is a disadvantage that the analysis accuracy is reduced due to loss due to or contamination of components unnecessary for analysis. Therefore, an expensive automatic dispensing / analyzing instrument is necessary to make up for such drawbacks.

  Therefore, in recent years, microfabrication technology can be applied to solve these problems, and flow channels can be formed and arranged on a chip with a size of several centimeters, and body fluid such as blood of a subject can be injected and analyzed there. New devices are being developed. As a function of such a device, a means for separating a specific component from a biological sample such as blood cell collection from blood has been demanded, and various techniques have been developed. For example, a separation technique using centrifugal force is known. For example, Patent Document 1 describes an apparatus for collecting blood cells from blood. Further, Patent Document 2 describes a chemical analyzer capable of quantitatively holding and supplying a liquid by centrifugal force by repeatedly rotating and stopping a chip having a channel disposed on a substantially horizontal plane. . Patent Document 3 discloses a technique in which a blood cell is separated from blood by rotating a chip having a flow path arranged in a substantially horizontal plane, and after stopping the rotation, a plasma component is separated using an external suction pump. Yes.

JP-A-3-270748 JP 2004-2112050 A Japanese Patent No. 3803078

However, the chip of Patent Document 1 is a chip having a valve mechanism composed of a high-precision spring and fine balls, and requires a plurality of precision parts. Therefore, in the microanalysis field where simple, quick and inexpensive analysis is required. Cost reduction is difficult. In addition, since the design is precise, there is a problem that skill is required for assembly. In addition, the chip of Patent Document 2 stops the chip after the serum separation operation, and guides the serum to the downstream mixing unit by capillary flow, but in an actual clinical sample, various components are mixed in the serum. Depending on the condition of the specimen, the components differ greatly, and there is a problem that variations occur in the amount and speed of capillary flow.
In the chip of Patent Document 3, after separation of blood cells, an externally connected suction pump is connected to collect plasma components. However, an externally connected pump is required. It was enough.
An object of the present invention is to provide a separation means for efficiently separating insoluble components such as blood cells from a suspension such as blood with a small device.

The present invention provides the following inventions.
[1] The chip is sequentially rotated at a first rotation speed and a second rotation speed higher than the first rotation speed with respect to a rotation axis outside the chip, and insoluble components are removed from the suspension by centrifugal force and gravity. A chip for separating, the first suspension tank for storing the introduced suspension and retaining insoluble components separated from the suspension by centrifugal force and gravity, the centrifugal force and A separation channel for causing components other than the insoluble components in the suspension separated by gravity to flow out, and the separation channel is connected to the first liquid storage tank and is located above the connection position. And a separation chip characterized by extending toward the inner peripheral side from a direction perpendicular to the resultant force of centrifugal force and gravity at the first rotational speed.
[2] The first liquid storage tank includes, in part, a holding region that is formed to protrude toward the outer peripheral side with respect to the rotation axis of the chip from the connection portion of the separation channel. Separation chip.
[3] A connecting portion between the separation channel and the first liquid storage tank is located on an inner peripheral side with respect to an interface between the insoluble component and the component other than the insoluble component at the first rotational speed. The separation chip according to [1] or [2].
[4] Any one of [1] to [3], wherein a part of the separation channel is located on an inner peripheral side of a plane including the liquid level of the suspension at the first rotational speed. Separation chip according to.
[5] The separation chip according to any one of [1] to [4], wherein the separation channel extends to an outer peripheral side with respect to a liquid level of the suspension at the second rotation speed.
[6] The separation chip according to any one of [1] to [5], wherein an angle of the separation channel with respect to a rotation axis is 27 ° or less.
[7] The separation chip according to any one of [1] to [6], in which the separation channel includes a trap projecting on the outer peripheral side of the separation channel.
[8] A second liquid storage tank for holding a component of the suspension other than the insoluble component is provided, and the second liquid storage tank is connected to the first liquid storage tank via the separation channel. The separation chip according to any one of [1] to [7], which communicates.
[9] The separation chip according to [8], having a drainage channel that connects the second liquid storage tank and the separation channel, and the drainage channel is connected to the separation channel. .
[10] The suspension is selected from blood, urine, spinal fluid, saliva, sputum, cell suspension, an enzyme reaction solution containing a carrier, an antigen-antibody reaction solution containing a carrier, and a reagent solution containing a carrier. The separation chip according to any one of [1] to [9].
[11] The separation chip according to any one of [1] to [10], wherein the suspension is blood and the insoluble component is blood cells.
[12] The separation chip according to any one of [1] to [10], wherein the suspension is a reagent including a carrier, and the insoluble component is a carrier.
[13] A method for separating an insoluble component from a suspension using the separation chip according to any one of [1] to [12], including the following steps.
(A) The step of introducing the suspension into the first storage tank of the separation chip (B) The separation chip is insoluble from the suspension by rotating the separation chip at the first rotation speed with respect to the rotation shaft outside the chip. Step of separating components (C) The separation chip is rotated at a second rotation speed higher than the first rotation speed with respect to the rotation shaft outside the chip to separate suspension components other than the insoluble components. [14] The separation method according to [13], wherein the centrifugal force obtained at the first rotational speed is 2G to 30G.
[15] The first rotation speed is such that the inclination of the liquid surface in the first liquid storage tank in the separation chip during rotation has an angle of 2 to 27 ° on the outer peripheral side with respect to the rotation axis. The separation method according to [13] or [14], wherein the separation speed is high.
[16] The separation method according to any one of [13] to [15], wherein the centrifugal force obtained at the second rotational speed is three times or more larger than the centrifugal force obtained at the first rotational speed.
[17] The separation method according to any one of [13] to [16], wherein the centrifugal force obtained at the second rotational speed is 50 G or more.
[18] The second rotation speed is such that the inclination of the liquid surface in the first liquid storage tank in the separation chip during rotation has an angle of 0 to 2 ° on the outer peripheral side with respect to the rotation axis. The separation method according to any one of [13] to [17], which is a speed.
[19] The suspension is selected from blood, blood cells, urine, spinal fluid, saliva, sputum, cell suspension, an enzyme reaction solution containing a carrier, an antigen-antibody reaction solution containing a carrier, and a reagent solution containing a carrier. The separation method according to any one of [13] to [18].
[20] The separation method according to any one of [13] to [19], wherein the suspension is blood and the insoluble component is blood cells.
[21] The separation method according to any one of [13] to [19], wherein the suspension is a reagent solution containing a carrier, and the insoluble component is a carrier.

  According to the present invention, by utilizing centrifugal force and gravity acting during rotation, components other than insoluble components can be efficiently separated from the suspension in a short time, and separation using the chips A method is provided.

(Separation chip of the present invention)
The separation chip of the present invention is a chip that sequentially rotates at a first rotation speed and a second rotation speed with respect to a rotation shaft outside the chip, and separates insoluble components from the suspension by centrifugal force and gravity.

  The separation chip of the present invention is used by rotating around the rotation shaft outside the chip. In the present invention, rotation means turning around a certain center axis (rotation axis), and is sometimes referred to as revolution with respect to rotation. The rotation trajectory may be substantially circular, and the trajectory radius is not particularly limited. The direction of the tip during rotation is usually such that the main surface (the surface in which the first liquid storage tank and the separation channel can be observed in the horizontal cross section) is oriented in the circumferential direction of the rotating track. In particular, when a suspension channel is provided, the suspension channel is tilted so that the suspension inlet is on the side of the rotation axis from the viewpoint of preventing suspension outflow from the rotating tip. It is preferable to rotate in a state. For example, as in the separation chip A of FIG. 1, the main surface is directed in the circumferential direction of the rotation orbit in a state where the suspension flow path 13 is inclined so that the first liquid storage tank 10 is positioned at the lowest position. Rotate. The inclination of the suspension channel can be set to a position that forms 10 to 80 °, preferably 20 to 50 ° with respect to the rotation axis.

  The shape of the separation chip of the present invention is usually a thin plate having a cubic or rectangular parallelepiped main surface. The size of the separation chip of the present invention may be any size that can be set in a centrifuge.

  The application target of the separation chip of the present invention is a suspension. The suspension may be a liquid in which a substance to be separated (whether solid or liquid) is mixed, and among them, a mixed liquid of biological components is preferable. For example, liquids collected from a living body including blood, urine, spinal fluid, saliva, sputum, cell suspension and the like can be mentioned. Further, an enzyme reaction solution containing a carrier, an antigen-antibody reaction solution containing a carrier, and a reagent solution containing a carrier may be used. Among these, a reagent solution containing blood or a carrier is preferable, and blood is most preferable. By rotating the suspension with a separation chip, insoluble components contained in the suspension can be separated. An insoluble component is a component that separates from the other components of the suspension when the suspension is rotated at the first rotational speed. For example, when the suspension is blood, it is a blood cell, and is a cell suspension. In the case of a liquid, it is various cells, in the case of an enzyme reaction solution containing a carrier, it is a carrier, in the case of an antigen-antibody reaction solution containing a carrier, it is a carrier, and in the case of a reagent solution containing a carrier, it is a carrier. is there. Among these, the separation chip of the present invention is most preferably used to separate blood cells (various blood cells such as red blood cells, white blood cells, and platelets) from blood as a suspension. It may also be used to separate a carrier from a reagent solution containing the carrier as a suspension.

  As described above, the separation chip of the present invention may be used for separating a carrier from an enzyme reaction solution containing a carrier, a carrier from an antigen-antibody reaction solution containing a carrier, and a carrier from a reagent solution containing the carrier. Good. Thereby, the test substance in the sample in the enzyme reaction solution containing the carrier, the antigen-antibody reaction solution containing the carrier, and the reagent solution containing the carrier can be analyzed.

  The test substance in the present invention may be a protein, sugar, lipid, nucleic acid, glycoprotein, glycolipid, or the like. Examples include cytokines, chemokines, interleukins, allergens, DNA, RNA, antibodies, lipids, enzymes, and other chemical substances. In particular, the test substance in the present invention is preferably a protein that binds to an antigen or an antibody, more preferably a cytokine / chemokine. More preferred are IL-6, IL-8, and TNF. The organism from which the test substance is derived does not matter. The test substance may be one type or two or more types.

  The specimen refers to a sample that may contain the test substance, and is preferably a liquid. For example, liquids collected from living bodies including body fluids such as blood, urine, spinal fluid, saliva, sputum, and cell suspension can be exemplified.

  The antigen-antibody reaction solution in the present invention is a solution containing an antibody or an antigen, or both an antibody and an antigen.

  The reagent of the reagent solution in the present invention means a chemical substance or a drug. For example, a drug or substance for detection or washing of a test substance, and more specifically, a labeled antibody (secondary antibody) labeled with fluorescence or an enzyme, an antigen, a binding protein, a fluorescent substrate, A cleaning liquid etc. can be mentioned. In addition, the reagent may be various chemical substances or drugs used in immunological measurement. Examples of the reagent solution in this case include a solution containing an antigen, a solution containing an antibody, a solution containing a washing solution, and an enzyme. It may be a solution containing a substrate, a specimen solution, or the like.

  The reagent solution in the present invention specifically includes a blocking solution, a diluent, a denaturing agent, a labeled antibody, a labeled antigen, an unlabeled antibody, an unlabeled antigen, a labeled substance, a luminescent substrate, a fluorescence in addition to a specimen. Substrate, chromogenic substrate, hydrogen peroxide solution, washing solution, protein denaturant, cell lysate, enzyme solution, labeled nucleic acid, unlabeled nucleic acid, primer, probe, avidin, streptavidin, enzyme solution, buffer solution, pH adjustment solution, high Examples include a solution containing a solution selected from a hybridization solution, an enzyme reaction stop solution and the like.

  The shape of the carrier in the present invention may be a so-called microrod such as a cylinder or a polygonal column, or a plate-like microplate, in addition to spherical or elliptical microbeads.

  The size of the carrier depends on the size of the channel including the reaction chamber and the separation channel, but the minor axis is preferably in the range of 1 to 1000 μm, preferably 10 to 200 μm, regardless of the shape of the carrier.

  The material of the carrier is not particularly limited, and glass, ceramic (eg, yttrium partially stabilized zirconia), metal (eg, gold, platinum, stainless steel), resin (eg, nylon, polystyrene, polyvinyl alcohol, polymethyl methacrylate, polyacrylamide), agarose Of these, resins, particularly polystyrene, are preferred.

  The shape, size and material of each carrier may be uniform or various. Moreover, it is not necessary that the antigen and / or antibody be bound to all of the carriers, and a portion of the carrier that does not bind anything may be included.

  In the enzyme reaction solution containing the carrier, the antigen-antibody reaction solution containing the carrier, and the reagent solution containing the carrier in the present invention, the carrier is preferably a carrier to which the antigen and / or antibody is bound. Antigens and / or antibodies to be bound to the carrier can be selected from various antibodies, antigen-binding fragments of antibodies such as Fab fragments and F (ab ′) 2 fragments, various antigens, and the like in samples in immunoassays. Antigens and antibodies that specifically bind to the test substance can be appropriately selected, and may be one kind or plural. There are no particular restrictions on the binding density, number of bindings, binding mode, etc. of the antigen or antibody to the carrier.

  The antigen and / or antibody can be bound to the carrier by, for example, a method in which the carrier and the antigen or antibody are mixed in a solution such as a buffer and contacted to bind. Bonding by contact can be carried out usually under conditions of 1 hour to 24 hours (days) at a low temperature, generally 4 to 37 ° C., with stirring as necessary. The obtained carrier may be washed with a buffer solution, a washing solution or the like before use. The binding method is not limited to this. For example, a method of chemically binding an antigen or antibody and a carrier using a crosslinking agent containing a hydrophilic polymer (polyethyleneimine, polyethylene glycol, polyvinyl alcohol, sodium polysulfonate, etc.). Etc. can also be used.

  Thus, by applying an enzyme reaction solution containing a carrier, an antigen-antibody reaction solution containing a carrier, and a reagent solution containing a carrier to the separation chip of the present invention, it is suspended in the enzyme reaction solution, reagent or antigen-antibody reaction solution. From the turbid and dispersed suspension, the carrier (insoluble component) and components other than the insoluble component can be separated.

  At this time, by increasing the amount of the carrier, a surface area larger than the immunological measurement using the plate can be used, and the reaction time can be shortened. Furthermore, when performing optical detection, if the suspension containing the carrier is left as it is, the presence of the carrier at the detection position causes light scattering, absorption, and autofluorescence by the carrier, resulting in measurement variations and noise. However, by using the separation chip of the present invention to separate the carrier and the solution and performing detection using only the solution, variation can be suppressed or a noise source can be eliminated.

  The separation chip of the present invention will be described below with reference to the drawings. FIGS. 1-9 is sectional drawing which looked at the isolation | separation chip | tip of each Example of this invention from the main surface side. 1 to 9 are separation chips each having a rotating shaft on the left side of the chip. Further, FIGS. 1 to 3 and 6 are particularly shown in a state when viewed from the circumferential direction of the track when the separation tip is actually rotated.

  The separation chip of the present invention has a first liquid storage tank and a separation channel. The first liquid storage tank is a liquid tank for storing the introduced suspension and holding insoluble components separated from the suspension by centrifugal force and gravity. The components other than the insoluble components in the suspension are mainly caused to flow out by centrifugal force and gravity when the chip is rotated at the second rotational speed.

  The size of the first liquid storage tank only needs to have a sufficient volume for storing the suspension, and may be, for example, a capacity capable of storing 20 μL to 50 mL of the suspension. The first liquid storage tank is connected directly or indirectly to the opening for injecting the suspension into the first liquid storage tank. From the viewpoint of prevention, operability at the time of solution injection, and the like, it is preferable to be connected to the opening via a suspension channel.

  The first liquid storage tank separates and holds insoluble components of the suspension by centrifugal force and gravity. That is, the suspension introduced into the first liquid storage tank separates insoluble components by the action of centrifugal force and gravity as the chip rotates, and this is held in a part of the first liquid storage tank. The For this reason, it is desirable that the first liquid storage tank includes a holding region for holding an insoluble component in a part thereof. The position of the holding region is formed so as to protrude to the outer peripheral side with respect to the rotation axis of the chip from the connection portion of the separation flow path, in other words, to the pressure direction side due to centrifugal force and gravity when the separation chip rotates. May be.

  In the separation chip A of FIG. 1, the first liquid storage tank 10 is provided below the rotation axis side of the chip as viewed from the main surface. The holding region 11 is formed so as to protrude to the outer peripheral side with respect to the rotation shaft.

  On the other hand, the separation flow path is connected to the first liquid storage tank described above, upward from the connection position, and on the inner peripheral side from the direction perpendicular to the resultant force of the centrifugal force and gravity at the first rotational speed. Stretch to. Thus, at the first rotation speed, the insoluble component in the suspension can be separated using the resultant force of centrifugal force and gravity without flowing out of the separation channel. In the separation chip A of FIG. 1, the separation flow path 20 opens from the connection portion 21 to the first liquid storage tank located at the lower part on the rotation axis side of the chip to the outer peripheral edge 22 obliquely above the chip.

  The connection portion between the separation channel and the first liquid storage tank is preferably located on the inner peripheral side with respect to the interface between the insoluble component and the component other than the insoluble component at the first rotational speed. Thereby, only components other than insoluble components can be efficiently separated from the suspension.

  The interface between the insoluble component and the component other than the insoluble component at the first rotation speed is due to the centrifugal force and gravity applied to the separation chip when the separation chip of the present invention is rotated at the first rotation speed. It is separated into an insoluble component separated from the suspension and a component other than the insoluble component in the suspension, which means an interface between the two layers. The insoluble component of the suspension forms the lower layer, and components other than the insoluble component form the upper layer, so that the interface is formed therebetween. Note that the timing at which the interface between the insoluble component and the other components is formed differs depending on the concentration and specific gravity of the insoluble component in the suspension and the type of the suspension, and usually when the first rotational speed is reached. Is formed, but it may be formed after a while after the first rotational speed is reached.

  When the separation chip of the present invention rotates, the connection portion between the separation channel and the first liquid storage tank is closer to the rotation axis than the interface. Means to be located in

  As shown in the separation chip G in FIG. 8 (an example when rotated at the first rotational speed), the connection portion 21 of the separation channel to the first liquid storage tank is in the first liquid storage tank 10. In the suspension of the point part of the chip, it is located closer to the rotation axis of the chip than the interface between the components other than the insoluble component of the upper layer (light colored part) and the insoluble component of the lower layer (dark colored part), and is insoluble Only components other than the components can flow into the separation channel by centrifugal force and gravity.

  Further, it is desirable that a part of the separation channel is located on the inner peripheral side with respect to the plane including the liquid level of the suspension at the first rotational speed. Thus, at the first rotation speed, the suspension and the insoluble component in the suspension can be efficiently separated without causing leakage from the flow path by utilizing the resultant force of centrifugal force and gravity.

The liquid level of the suspension at the first rotational speed is the liquid that the suspension forms in the first liquid storage tank or suspension channel when the separation chip is rotated at the first rotational speed. Means a face. The plane including the liquid level of the suspension means the liquid level as it is when the liquid level is flat, and means the tangent of the liquid level at the center of the flow path when the liquid level is not flat. To do.
The liquid that enters the flow path is a suspension at a point in time immediately after starting rotation at the first rotation speed, but as the insoluble component is separated by rotation at the first rotation speed, The ratio of components other than insoluble components in the liquid gradually increases, and in some cases, only components other than insoluble components may be present.

  A part of the separation channel is located on the inner peripheral side of the plane including the liquid level of the suspension. When the separation chip of the present invention rotates at the first rotation speed, the separation channel Part of the separation channel, for example, the portion from the connection point between the separation channel and the first liquid storage tank to the intermediate point of the separation channel (intermediate point between the connection part and the end point of the channel) to the latter half is from the plane. Is also located on the rotating shaft side. In other words, when observed from the main surface of the chip, it means that components other than the suspension or the insoluble component have flowed only halfway through the separation channel and have not reached the end point.

  The separation channel 20 of the separation chip A of FIG. 1, the separation chip B of FIG. 2, and the separation chip C of FIG. 3 have a rotational axis that is more rotational than the liquid level (1) during rotation at the first rotational speed. It is located on the inner circumference side. That is, the inclination of the separation channel 20 is larger than the inclination of the liquid level (1), and the liquid level (1) reaches the point a in the middle of the separation channel 20, so the separation channel No components other than the suspension or insoluble components will overflow from the end point.

  Further, it is desirable that the separation channel extends to the outer peripheral side with respect to the liquid level of the suspension at the second rotational speed. Thereby, components other than the insoluble component separated in the rotation at the first rotation speed can be reliably recovered by the rotation at the second rotation speed.

  The liquid level of the suspension at the second rotational speed refers to the suspension (usually separated from insoluble components at the first rotational speed when the separation chip is rotated at the second rotational speed. In addition, it means a liquid surface formed in the first liquid storage tank or the suspension flow path by a component other than the insoluble component of the suspension. Further, the plane including the liquid level means the liquid level as it is when the liquid level is flat, and when the liquid level is other than the plane, the tangent of the liquid level at the center of the flow path is the same as described above. Means.

  The separation flow path extends to the outer peripheral side from the liquid level of the suspension at the second rotation speed. When the separation chip of the present invention rotates at the second rotation speed, the separation flow The liquid surface is positioned closer to the rotating shaft than a part of the path, for example, a part other than the connection part between the separation channel and the first liquid storage tank, preferably a part including the end point of the separation channel. Means. In other words, when observed from the main surface of the chip, it means that the suspension, that is, the components other than the insoluble component, has reached the end point of the separation channel.

The separation flow path 20 of each of the separation chip A in FIG. 1, the separation chip B in FIG. 2, and the separation chip C in FIG. 3 extends to the outer peripheral side from the liquid surface (2) during rotation at the second rotational speed. ing. That is, the inclination of the separation channel 20 is smaller than the inclination of the liquid level (2) with respect to the horizontal plane. That is, since the liquid level (2) reaches the end point 22 of the separation channel 20, components other than the insoluble component can be taken out from the end point of the separation channel.
The components other than the insoluble components taken out from the suspension do not need to be the total amount of the components other than the insoluble components, and only a part of them can be taken out.

As described above, the separation channel extends upward from the connection position with the first liquid storage tank, and extends inward from the direction perpendicular to the resultant force of centrifugal force and gravity at the first rotational speed. Although it is necessary, it is preferable to extend in the outer peripheral direction slightly from the rotation axis of the separation chip. Specifically, the angle of the separation channel with respect to the rotation axis of the separation chip can be determined according to the centrifugal force at the first rotational speed that is actually used, and is the direction of the resultant force of the centrifugal force and gravity. It is preferable. For example, when a centrifugal force of 2 G or less is applied at the first rotation speed, the angle of the separation channel with respect to the rotation axis of the separation chip is preferably 27 ° or less. In addition, when a centrifugal force of 5 G or less is applied at the first rotation speed, the angle of the separation channel with respect to the rotation axis of the separation chip is preferably 12 ° or less. Furthermore, when a centrifugal force of 10 G or less is applied at the first rotation speed, the angle of the separation channel with respect to the rotation axis of the separation chip is preferably 6 ° or less. Furthermore, when a centrifugal force of 30 G or less is applied at the first rotational speed, the angle of the separation channel with respect to the rotation axis of the separation chip is preferably 2 ° or less. More preferably, the angle of the separation channel with respect to the rotation axis is 0 to 5 °, and more preferably 1 to 2 °.
The separation flow path 20 of each of the separation chip A in FIG. 1, the separation chip B in FIG. 2, and the separation chip C in FIG. 3 is very slightly with respect to the rotation axis of the separation chip (perpendicular to the separation chip in the figure). It extends toward the outer periphery.

  At the end of the separation channel on the outer peripheral side, it is preferable to connect a second liquid storage tank as will be described later, since it becomes easy to collect components other than the insoluble components of the suspension. On the other hand, in the separation chip B of FIG. 2 and the separation chip C of FIG. 3, the separation flow path 20 goes obliquely upward from the connection portion 21 but communicates with the opening 31 and the second storage tank 30. Connect to the drainage flow path 32.

  The separation flow path has a trap protruding in the middle of the separation flow path to adjust the flow in the separation flow path, and at the same time, captures the unnecessary components slightly mixed in the separation flow path. It is preferable because it can be prevented from flowing out to the second storage tank downstream. The trap can be provided at an arbitrary position in the middle of the separation channel, preferably at the latter half. For example, in the separation chip D of FIG. 4, the trap 23 is provided on the side of the separation channel 20 close to the connection part to the drainage channel 32.

  The shape and size of the separation channel need only be tube-shaped as a whole and may not be constant throughout the separation channel. The shape of the cross section is not particularly limited, such as a circle or a polygon. The size of the cross section may be approximately constant, and can be adjusted as appropriate so that components other than the insoluble components of the suspension can pass through. In order to prevent the suspension from entering the separation channel before tip rotation due to capillary action, it is preferable to secure a certain size of the cross section. For example, the short diameter (in the case of a circle, the diameter means the shortest diameter passing through the center in the case of a polygon) is usually 10 μm to 10 mm, preferably 500 μm to 5 mm.

  In addition, the separation flow path does not necessarily have to be a straight line, and a part or all of the separation flow path may draw a curve or an unevenness. For example, as shown in the separation chip E of FIG. 5, a part 24 on the second storage tank side of the separation channel 20 may draw a curve. Further, as shown in the separation chip F in FIG. 6, the separation channel 20 may be bent at the end portion 25.

  Thus, even when the entire separation channel is not a straight line, as described above, the connection portion between the separation channel and the first liquid storage tank has the insoluble component and the insoluble component at the first rotational speed. Located on the inner peripheral side of the interface with the other components, a part of the separation channel being located on the inner peripheral side than the plane including the liquid level of the suspension at the first rotational speed, and Needless to say, each of the separation channels is preferably extended to the outer peripheral side of the liquid surface of the suspension at the second rotational speed. Specifically, the angle formed by the straight line and the rotation axis of the separation chip is based on a straight line connecting the portion of the separation flow channel closest to the rotation axis and the connection portion of the first liquid storage tank. The above-mentioned predetermined angle is preferable.

  The opening of the first liquid storage tank can be provided directly in the first liquid storage tank, but the opening for injecting the suspension, the opening, and the first liquid storage tank By having the suspension flow path connecting the two, the suspension can be easily introduced into the first liquid storage tank.

  The suspension channel can be provided at a position that does not hinder the inflow of components into the separation channel. For example, in the first liquid storage tank, the suspension channel is 10 to 80 °, preferably 20 °. It can be provided to intersect at an angle of ~ 60 °. Further, when the chip outer shape is a rectangular parallelepiped (rectangular), the suspension flow path can be provided so as to be a straight line substantially parallel to the edge on the rotation axis side of the chip.

  The shape and size of the suspension channel need only be tube-shaped as a whole and may not be constant. The shape of the cross section is not particularly limited, such as a circle or a polygon. The size of the cross section may be approximately constant, but the suspension channel is compared with the separation channel from the viewpoint of facilitating introduction of the suspension and recovery of insoluble components after rotation of the separation chip. Therefore, it is preferable to take a wide width. For example, the short diameter (in the case of a circle, the diameter means the shortest diameter passing through the center in the case of a polygon) is usually in the range of 1 mm to 20 mm, preferably 3 mm to 9 mm.

  In the separation chips A to E of the embodiments of FIGS. 1 to 5, the suspension channel 13 is provided in a straight line substantially parallel to the edge on the rotation axis side of the chip. The suspension flow path 13 is connected to the first liquid storage tank 10 and intersects the separation flow path 20 at the connection portion.

  On the other hand, the suspension inlet may be provided directly in the first liquid storage tank without providing the suspension flow path. The suspension inlet is preferably provided in a portion where the liquid level of the suspension of the first liquid storage tank does not reach from the viewpoint of preventing leakage of the suspension. Specifically, the first liquid storage tank can be provided on the inner peripheral side with respect to the rotation axis of the chip and vertically above the rotation axis of the chip with respect to the connection portion of the separation channel.

  In the separation chip F shown in FIG. 6, a suspension inlet 14 is provided in the first liquid storage tank 10. The suspension inlet 14 is located above the chip and is more than the liquid level (1) of the suspension at the first rotational speed and the liquid level (2) of the suspension at the second rotational speed. Located on the inner circumference.

In the separation chip of the present invention, it is preferable to have the second liquid storage tank for holding the components of the suspension other than the insoluble components because the recovery becomes easy. The second liquid storage tank communicates with the first liquid storage tank via the separation channel. The second liquid storage tank can be located at the end of the separation channel.
For example, in the separation chip B of FIG. 2, the second liquid storage tank 30 is connected to the final end of the separation channel 20. Also in the separation chip F of FIG. 6, the end portion 25 of the separation flow path 20 is bent, and a second liquid storage tank 30 is provided at the end thereof. An opening for collecting components other than the insoluble components is provided at an arbitrary portion.

In addition, the second liquid storage tank and the separation channel may be connected by a drainage channel, whereby the suspension components other than the insoluble component when the tip rotation is stopped. , Back flow to the separation channel can be prevented.
For example, in the separation chip C of FIG. 3, the drainage channel 32 is connected to the upper right end of the separation channel 20. The drainage channel 32 extends from the top to the bottom of the chip substantially parallel to the suspension channel 13. A second liquid storage tank 30 is provided at the lower end of the drainage flow path 32, and an opening 31 is provided at the upper end.

  The connecting portion between the separation channel and the drainage channel preferably forms an angle of 10 to 80 °. As shown in FIG. 3, the drainage channel can be positioned substantially parallel to the suspension channel. When the drainage flow path is provided, the second liquid storage tank can be positioned at the corner portion of the edge farther from the rotation axis of the separation chip, like the separation chip D of FIG.

  The material of the separation chip of the present invention is preferably a transparent material because observation can be performed from the outside. Transparent materials include polymethyl methyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethylsiloxane, polyethylene terephthalate, cycloolefin polymer, fluororesin, silicon, etc. Resins, copolymers or composites containing such polymer compounds; glasses such as quartz glass, Pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate glass, and composites thereof; A metal whose surface is coated with an insulating material and a composite thereof, ceramics and a composite thereof are preferably used. Of these, polymethyl methyl methacrylate (PMMA), polypropylene, polystyrene, and polycarbonate are particularly preferably used.

  The manufacturing method of the separation chip of the present invention is not particularly limited. For example, a plate-like substrate on which each liquid storage tank and a concave portion of each flow path are formed can be bonded to another substrate or film. Alternatively, it can be created by sandwiching a substrate having a slit forming a flow path between two substrates from both sides. The formation of the recesses in each liquid storage tank and each flow path is a general molding method using a mold when the material is resin, for example, injection molding, extrusion molding, blow molding, vacuum molding, hot embossing. And so on.

(Separation method of the present invention)
The separation method of the present invention is a method for separating an insoluble component from a suspension using the above-described separation chip, and includes the following steps.
(A) The step of introducing the suspension into the first storage tank of the separation chip (B) The separation chip is insoluble from the suspension by rotating the separation chip at the first rotation speed with respect to the rotation shaft outside the chip. Step of separating components (C) The separation chip is rotated at a second rotation speed higher than the first rotation speed with respect to the rotation shaft outside the chip to separate suspension components other than the insoluble components. Discharging from the first storage tank via

  The separation chip and suspension in the separation method of the present invention are as already described in the section of the separation chip of the present invention.

  First, in step (A), the suspension is introduced into the first storage tank of the separation chip. The suspension can be introduced according to a conventional method using a pipette or the like. In the step (A), as in the separation chip G of FIG. 7, the suspension is introduced from the opening 12 through the suspension flow path 13 and includes the holding region 11 of the first liquid storage tank 10. The suspension (shaded area in the figure) is stored.

  In the step (B), the separation chip is revolved at the first rotation speed with respect to the rotation shaft outside the chip to separate insoluble components from the suspension. The definitions of the rotation axis and revolution are as described in the description of the separation chip of the present invention. In step (B), the centrifuge is usually set to the centrifuge so that the angle of the separation chip G shown in FIG. 8 is seen from the horizontal direction, and the separation chip G revolves around the left side as the rotation axis at the first rotation speed. Begin centrifugation.

The centrifugal force obtained by the first rotation speed is usually 2 to 30 G, and preferably 8 to 15 G.
Further, the first rotation speed is such that the inclination of the liquid surface in the first liquid storage tank in the separation chip during rotation is normally 2 to 27 ° on the outer peripheral side with respect to the rotation axis (vertical axis) of the chip. The speed is preferably 2 to 12 °, more preferably 5 to 10 °. This angle can also be defined so that the inclination of the liquid surface in the suspension channel falls within the above range when a chip having a suspension channel is used as the separation chip.

  Further, the first rotation speed is such that the inclination of the liquid level in the separation channel in the separation chip during rotation is generally 1 to 11 °, preferably 3 to 6 ° larger than that of the separation channel. You may set suitably.

  In the present invention, the liquid level when the channel width is sufficiently wide, such as the first liquid storage tank or the suspension channel, is a portion where the liquid level is raised by the surface tension on the wall surface (in the case of hydrophilicity) ) Rather than a flat surface near the center of the flow path. On the other hand, the liquid level of the separation channel is that the separation channel has a narrow channel width, so that the portion where the liquid level is raised by the surface tension is connected to the wall surface opposite to the channel wall surface. Since such a flat portion cannot be formed, the tangent of the liquid level at the center of the flow path is defined as the liquid level.

  The rotation time at the first rotation speed can be 1 to 30 minutes, preferably 5 to 20 minutes.

  (B) If the example of a process is given, it will set to the centrifuge in the direction of the isolation | separation chip | tip G of FIG. 8, and it will rotate about 20 minutes with the centrifugal force 10G. During the rotation of the chip, insoluble components are accumulated in the holding region 11 of the first liquid storage tank 10, and the suspension (which may contain insoluble components in this step) is separated. 20 is pushed up and the liquid level reaches the line (1). Since the liquid level reaches only the line (1), it does not reach the drainage flow path 32. (B) At the end of the process (at the end of rotation at the first rotation speed), the insoluble component in the suspension is accumulated in the holding region 11.

  In the step (C), the separation chip is rotated at the second rotation speed with respect to the rotation shaft outside the chip, and the suspension components other than the insoluble components are discharged from the first liquid storage tank through the separation channel. To do.

  The second rotational speed needs to be higher than the first rotational speed, and is preferably three times or more larger than the centrifugal force obtained at the first rotational speed. Specifically, the speed is preferably 50 to 3000 G higher than the centrifugal force obtained at the first rotation speed, and more preferably 70 to 1000 G high speed. The specific range of the second rotation speed is usually 50G or more, preferably 100G or more. Especially preferably, it is 50-3000G, More preferably, it is 100-1000G.

Further, the second rotation speed is such that the inclination of the liquid surface in the first liquid storage tank in the separation chip during rotation is normally 0 to 2 ° on the outer peripheral side with respect to the rotation axis (vertical axis) of the chip. The speed is preferably such that it has an angle of 0 to 1 °. This angle can also be defined so that the inclination of the liquid surface in the suspension channel falls within the above range when a chip having a suspension channel is used as the separation chip.
When 30 G is given as the second rotation speed, the inclination of the liquid level in the first liquid storage tank in the separation chip during rotation is approximately 2 ° on the outer peripheral side with respect to the rotation axis (vertical axis) of the chip. It becomes. In addition, when 50 G is given as the second rotation speed, the inclination of the liquid level in the first liquid storage tank in the separation chip during rotation is approximately on the outer peripheral side with respect to the rotation axis (vertical axis) of the chip. 1 °.

  The rotation time at the second rotation speed can be 0.5 to 5 minutes, preferably 1 to 3 minutes.

  In the step (C), usually set in the centrifuge so as to be at the angle of the separation chip G in FIG. 9 when viewed from the horizontal direction (that is, in the state set in the centrifuge in the step (B)), Centrifugation is started so that the separation chip G revolves around the left side as the rotation axis at two rotation speeds. (C) If the example of a process is given, it will set to a centrifuge with the direction of the isolation | separation chip | tip G of FIG. 9, and will rotate about 2 minutes with the centrifugal force 100G. During the rotation of the chip, the insoluble component accumulated in the holding region 11 of the first liquid storage tank 10 does not move as it is, the component other than the insoluble component is pushed up the separation channel 20, and the liquid level is (2). The line reaches the drainage flow path 32 and is accumulated in the second liquid storage tank 30.

  The insoluble component accumulated in the holding region of the first liquid storage tank can be collected with a dropper or the like after the end of the step (C). In addition, the suspension other than the insoluble component can be collected with a dropper or the like after the completion of the step (C), or can be collected by turning the chip upside down.

  Although an example of implementation of the separation chip in the present invention is shown, the present invention is not limited to the example.

Example 1
A separation chip was manufactured as follows. The separation chip produced in this example is composed of a lower plate and a cover plate in which a tank and a flow path are formed. Each production method is as follows.

-Lower plate: Kuraray PMMA extrusion plate "Comoglas" A prototype lower plate of a separation chip was manufactured by cutting using a milling machine so that a thickness of 4 mm was designed as shown in FIG. The shunt depth was 0.5 mm, the trap depth was 1 mm, and the other depth was 3.5 mm.
Cover plate: A “como glass” thickness of 1 mm was cut into the same dimensions as the bottom plate to form a cover plate.
-Joining of lid plate and lower plate: The lid plate was placed so that the joint surface was facing upward. Methanol was pipetted with 100 to 200 μL, and the lid plate surface was immersed. The lower plate tank and the flow path surface (cutting surface) were overlapped on the cover plate so as to fit, and the cover plate side was pressed and bonded at 3 KN for 3 minutes with a hot press machine preheated to 60 ° C.

Example 2
100 μL of blood is introduced into the first storage tank (10) through the suspension channel (13) from the opening (12) of the separation chip manufactured in Example 1, and then the second storage of the separation chip. It inserted in the centrifuge so that the liquid tank 30 might become a centrifugal outer peripheral side. The separation chip is centrifuged for 20 minutes with a centrifugal force of 10 G to separate blood into plasma components and blood cell components in the first reservoir (10), and further centrifuged for 1 minute with a centrifugal force of 100 G to remove only the plasma components. The liquid was sent to the second liquid storage tank (30) via the separation channel (20).

  Table 1 shows the results of measuring the plasma components fed to the second reservoir 30 with a hemocytometer. From this result, it was possible to obtain a result that the plasma component can be separated from the blood and sent to the second storage tank by the operation of only the above-mentioned centrifugation.

Production Example 1
For immunological measurement by antigen-antibody reaction on the surface of the carrier, a suspension of a substrate solution containing beads as a carrier and labeled antibody as a reagent antibody was prepared.

  Polystyrene beads (Polyscience, particle size: 25 μm) are selected as a carrier, washed with a phosphate buffer, and the same amount of 0.1 μg / ml anti-hIL-6 antibody phosphate buffer as polystyrene beads is added. Penetration overnight at ° C. After infiltration, 10 μl of polystyrene beads were suspended in 150 μl of phosphate buffer containing 0.05% Tween 20.

  Thereafter, 50 μl of hIL-6 (human interleukin-6, Kamakura Technoscience) dissolved in a phosphate buffer containing 0.25% bovine serum albumin (BSA) and 0.05% Tween 20 and 0.25% A 0.1 μg / ml HRP (horseradish peroxidase) -labeled anti-hIL-6 antibody (Kamakura Technoscience) 50 μl dissolved in a phosphate buffer containing BSA and 0.05% Tween 20 was mixed and reacted for 120 seconds. It was. After the reaction, it was washed with 100 μl of a phosphate buffer containing 0.05% Tween 20 and then 100 μl of 0.1 M Tris-HCl buffer (pH 7.5) containing 200 μM hydrogen peroxide solution and 13 μg / ml Amplex Red (Molecular Probes). Was added as a substrate solution and allowed to react at room temperature for 5 minutes.

Example 3
100 μl of the suspension of the beads and the substrate solution prepared in Production Example 1 was introduced into the first liquid storage tank (10) through the suspension channel (13) from the opening (12) of the separation chip, and then separated. It inserted into the centrifuge so that the 2nd liquid storage tank (30) of a chip | tip might become a centrifugal outer peripheral side. Centrifuge at 10G for 5 minutes, separate into polystyrene beads and reaction solution in the first reservoir, and further centrifuge for 1 minute at 100G to transfer only the reaction solution to the second reservoir. Liquid was sent.

  The amount of resorufin in the reaction solution sent to the second liquid storage tank was measured using a fluorescence microscope IX-71 (Olympus). The measurement results of the fluorescence intensity of hIL-6 at each concentration are shown in FIG. (Excitation wavelength 510-560 nm, emission wavelength 575-650 nm, exposure time 0.5 seconds)

Comparative Example 1
After leaving 100 μl of the suspension of beads and substrate solution prepared in Production Example 1 for 6 minutes without using a separation chip, the amount of resorufin in the reaction solution was measured using a fluorescence microscope IX-71 (Olympus). It was measured. The measurement results of the fluorescence intensity of hIL-6 at each concentration are shown in FIG. (Excitation wavelength 510-560 nm, emission wavelength 575-650 nm, exposure time 0.5 seconds)

  In Example 3 and Comparative Example 1, calibration curves as shown in FIGS. 10 and 11 could be created. Here, compared with the calibration curve of FIG. 10, the slope of each calibration curve of FIG. 11 is small. Further, the variation 3SD at each measurement point in FIG. 11 is larger than that in FIG. From these, the following considerations are possible. That is, from the comparison of the measurement results of Example 3 and Comparative Example 1, it is possible to increase the concentration of resorufin per volume by rotating the separation chip as in Example 3 and eliminating the beads from the reaction solution. The inclination can be increased. In addition, it is possible to eliminate noise sources and variations caused by scattering and absorption of light derived from beads, autofluorescence, to reduce 3SD variation, and to obtain a result that detection in a low concentration range is possible. I was able to.

FIG. 1 is a cross-sectional view schematically showing an embodiment of the separation chip of the present invention. FIG. 2 is a cross-sectional view schematically showing an embodiment of the separation chip of the present invention. FIG. 3 is a sectional view schematically showing one embodiment of the separation chip of the present invention. FIG. 4 is a cross-sectional view schematically showing one embodiment of the separation chip of the present invention. FIG. 5 is a cross-sectional view schematically showing one embodiment of the separation chip of the present invention. FIG. 6 is a cross-sectional view schematically showing an embodiment of the separation chip of the present invention. FIG. 7 is an explanatory view schematically showing step (A) of the separation method of the present invention. FIG. 8 is an explanatory view schematically showing the step (B) of the separation method of the present invention. FIG. 9 is an explanatory view schematically showing the step (C) of the separation method of the present invention. FIG. 10 is a diagram showing measurement results obtained using the separation chip of the present invention. FIG. 11 is a diagram showing measurement results obtained without using a separation chip.

Explanation of symbols

A to G Separation chip 10 First liquid storage tank 11 Holding region 12 Opening section 13 Suspension flow path 14 Suspension introduction port 20 Separation flow path 21 Connection section 22 to the first liquid storage tank 22 Opening portion 23 Trap 24 Curve portion 25 End portion 30 of separation channel Second storage tank 31 Opening portion 32 of drainage channel Drainage channel

Claims (21)

In order to separate the insoluble component from the suspension by centrifugal force and gravity by sequentially rotating the chip at a first rotation speed and a second rotation speed higher than the first rotation speed with respect to the rotation axis outside the chip. Chips,
A first liquid storage tank for storing the introduced suspension and retaining insoluble components separated from the suspension by centrifugal force and gravity;
A separation flow path for causing components other than the insoluble components in the suspension separated by the centrifugal force and gravity to flow out,
The separation channel is connected to the first liquid storage tank, and extends upward from the connection position and extends inward from the direction perpendicular to the resultant force of centrifugal force and gravity at the first rotational speed. Separation chip characterized by The first liquid storage tank includes a holding region formed in a part thereof so as to protrude to the outer peripheral side with respect to the rotation axis of the chip from the connection portion of the separation channel.
The separation chip according to claim 1.   The connection part of the said separation flow path and the said 1st liquid storage tank is located in the inner peripheral side rather than the interface of the said insoluble component in said 1st rotational speed, and components other than an insoluble component. Or the separation chip | tip of 2.   The separation chip according to any one of claims 1 to 3, wherein a part of the separation channel is located on an inner peripheral side with respect to a plane including the liquid level of the suspension at the first rotation speed. .   The separation chip according to any one of claims 1 to 4, wherein the separation channel extends to an outer peripheral side with respect to a liquid level of the suspension at the second rotation speed.   The angle with respect to the rotating shaft of the said separation flow path is 27 degrees or less, The separation chip as described in any one of Claims 1-5 characterized by the above-mentioned. The separation flow path has a trap protruding on the outer peripheral side of the separation flow path in the middle thereof,
The separation chip according to any one of claims 1 to 6. A second reservoir for holding the components of the suspension other than the insoluble components;
The second storage tank communicates with the first storage tank via the separation channel;
The separation chip according to claim 1. A drainage channel connecting the second liquid storage tank and the separation channel;
The drainage channel is connected to the separation channel;
The separation chip according to claim 8.   The suspension is selected from blood, urine, spinal fluid, saliva, sputum, cell suspension, an enzyme reaction solution containing a carrier, an antigen-antibody reaction solution containing a carrier, and a reagent solution containing a carrier. The separation chip according to any one of?   The separation chip according to any one of claims 1 to 10, wherein the suspension is blood and the insoluble component is a blood cell.   The separation chip according to claim 1, wherein the suspension is a reagent including a carrier, and the insoluble component is a carrier. A method for separating an insoluble component from a suspension using the separation chip according to any one of claims 1 to 12, comprising the following steps.
(A) The step of introducing the suspension into the first storage tank of the separation chip (B) The separation chip is insoluble from the suspension by rotating the separation chip at the first rotation speed with respect to the rotation shaft outside the chip. Step of separating components (C) The separation chip is rotated at a second rotation speed higher than the first rotation speed with respect to the rotation shaft outside the chip to separate suspension components other than the insoluble components. Discharging from the first storage tank via   The separation method according to claim 13, wherein the centrifugal force obtained at the first rotation speed is 2G to 30G.   The first rotation speed is such that the inclination of the liquid surface in the first liquid storage tank in the separation chip during rotation has an angle of 2 to 27 ° on the outer peripheral side with respect to the rotation axis. The separation method according to claim 13 or 14, wherein:   The separation method according to any one of claims 13 to 15, wherein the centrifugal force obtained at the second rotational speed is three times or more larger than the centrifugal force obtained at the first rotational speed.   The separation method according to any one of claims 13 to 16, wherein the centrifugal force obtained at the second rotation speed is 50G or more.   The second rotation speed is such that the inclination of the liquid surface in the first liquid storage tank in the rotating separation chip has an angle of 0 to 2 ° on the outer peripheral side with respect to the rotation axis. The separation method according to any one of claims 13 to 17.   The suspension is selected from blood, blood cells, urine, spinal fluid, saliva, sputum, cell suspension, an enzyme reaction solution containing a carrier, an antigen-antibody reaction solution containing a carrier, and a reagent solution containing a carrier. Item 19. The separation method according to any one of Items 13 to 18.   The separation method according to any one of claims 13 to 19, wherein the suspension is blood and the insoluble component is blood cells.   The separation method according to any one of claims 13 to 19, wherein the suspension is a reagent containing a carrier, and the insoluble component is a carrier.

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