JP2011080769A - Disk-like analyzing chip and measuring system using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a disk-like analyzing chip capable of more strictly controlling the profile of multistage liquid sending by changing the centrifugal force caused by rotation, and to provide a simple measuring system using the chip. <P>SOLUTION: The disk-like analyzing chip is used in the measuring system for performing measurement by arranging the disk-like analyzing chip on a centrifugal device and utilizing the centrifugal force produced by the rotation of the centrifugal device to move the sample on the disk-like analyzing chip, before reacting the same with a reagent and the surface of the disk-like analyzing chip, is equipped with a sample tank; a reaction tank provided in the direction of the outer peripheral part of the disk-like analyzing chip with respect to the sample tank; a measuring tank provided in the direction of the outer peripheral part of the disk-like analyzing chip with respect to the sample tank; and a first flow channel connected to a sample introducing tank and the reaction tank and the second flow channel connected to the reaction tank and the measuring tank. The cross-sectional area of the first flow channel is larger than that of the second flow channel. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  In the present invention, the disk-type analysis chip is placed on a centrifuge such as a turntable, the sample on the disk-type analysis chip is moved using the centrifugal force generated by the rotation of the centrifuge and reacted with a reagent. The present invention relates to a measurement system that performs the above and a disk-type analysis chip used therefor.

  In recent years, the importance of detecting, detecting or quantifying biological substances such as DNA (Deoxyribo Nucleic Acid), enzymes, antigens, antibodies, proteins, viruses and cells, and chemical substances in fields such as medicine, health, food, and drug discovery Various analysis chips and microchemical chips (hereinafter collectively referred to as analysis chips) that can easily measure them have been proposed. The analysis chip allows a series of experiments and analysis operations performed in the laboratory to be performed within a chip of several cm square and a thickness of about several millimeters to 1 cm. There are many advantages such as a high reaction rate, high-throughput testing, and the ability to obtain test results immediately at the site where the sample is collected. Such an analysis chip is suitably used for biochemical tests such as blood tests.

  As an example of an analysis chip, a “fluid circuit” that is a flow channel network including a portion (chamber) for performing a specific process on a fluid and a fine flow channel that appropriately connects these portions inside the analysis chip. An analysis chip equipped with Using such an analysis chip having a fluid circuit inside, specimen inspection / analysis (for example, when the specimen is blood, measurement of blood or a specific component contained in blood can be mentioned). When performing, various fluid processes, such as measurement of the sample introduced into the fluid circuit and the reagent mixed with the fluid circuit, and mixing of the sample and the reagent, are performed using the fluid circuit. Such various fluid treatments can be performed by applying a centrifugal force in an appropriate direction to the analysis chip.

  Here, a so-called reagent built-in type analysis chip is known in which a sample or a reagent for mixing or reacting with a specific component in the sample is previously incorporated and held in a fluid circuit. A reagent built-in type analysis chip is usually provided with one or more reagent holding tanks for holding a reagent as part of its fluid circuit, and the reagent holding tank is filled and sealed when the analysis chip is manufactured. However, it is shipped for use in such a state.

  In recent years, the development of disk-type analysis chips for flowing specimens and beads in a fluid circuit using centrifugal force due to rotation has been promoted (Patent Documents 1 and 2), and a disk shape such as a compact disk (CD). A large number of reservoirs and microchannels connecting them are created on the plate of the plate, and the centrifugal force generated by rotating the reservoirs is used to sequentially flow the liquid in the reservoirs through the microchannels (multistage feeding). Liquid) has also been developed (Non-Patent Document 1). By using such a method, liquid can be sent without using peripheral equipment such as pumps and valves, so the entire analysis system can be miniaturized, and reagent addition and washing required for immunoassay methods, etc. There is an advantage that the complicated operation can be automated. In addition, since a minute space is used as a reaction field, even a very small amount of sample can be measured, and the analysis time can be shortened.

JP 2003-185671 A JP-A-2005-241453

Hideshi Nakajima, “Flow analysis method using compact disc microchip”, “Bunseki” Japan Society for Analytical Chemistry, July 2009, p. 381-382

  However, in the measurement method described in Patent Document 1, it is usually necessary to scan the whole by moving the point sensor unit in a linear direction along the flow path, and such a scanning device is required. In addition, in the disk-type analysis chip disclosed in Non-Patent Document 1, a solution is caused to flow in each reservoir by adjusting the capillary force of the junction portion of the fine channel and the centrifugal force due to rotation. In order to strictly control the holding time, a complicated circuit design is required. In addition, an error in the holding time (reaction time) in each reservoir may lead to a measurement error.

  Therefore, the present invention provides a disk-type analysis chip capable of more strictly controlling the profile of multistage liquid feeding by changing the centrifugal force due to rotation, and a simple measurement system using the same. Objective.

The present invention is a measurement in which a disk-type analysis chip is placed on a centrifuge, and the sample on the disk-type analysis chip is moved using the centrifugal force generated by the rotation of the centrifuge and reacted with a reagent. A disk-type analysis chip used in the system,
On the surface of the disk-type analysis chip, a sample tank, a reaction tank provided in the direction of the outer periphery of the disk-type analysis chip with respect to the sample tank, and provided in the direction of the outer periphery of the disk-type analysis chip with respect to the reaction tank A measurement tank, a first flow path connecting the sample introduction tank and the reaction tank, and a second flow path connecting the reaction tank and the measurement tank,
The disk-type analysis chip is characterized in that a cross-sectional area of the first flow path is larger than a cross-sectional area of the second flow path.

  It is preferable to have a water repellent coating layer on the inner surfaces of the first flow path and the second flow path.

  The measurement is measurement of the amount of fluorescence detected, and the material constituting the measurement tank is preferably a material that hardly generates fluorescence.

The material that hardly generates fluorescence is preferably a (meth) acrylic resin.
It is preferable that the reaction tank and the measurement tank have an exhaust port.

Further, the present invention is a measurement system including a centrifuge device and a disk-type analysis chip installed on the centrifuge device,
The sample on the disk-type analysis chip is moved using the centrifugal force generated by the rotation of the centrifuge, and the measurement is performed after reacting with the reagent.
On the surface of the disk-type analysis chip, a sample tank, a reaction tank provided in the direction of the outer periphery of the disk-type analysis chip with respect to the sample tank, and provided in the direction of the outer periphery of the disk-type analysis chip with respect to the reaction tank A measurement tank, a first flow path connecting the sample introduction tank and the reaction tank, and a second flow path connecting the reaction tank and the measurement tank,
The invention also relates to a measuring system, characterized in that the cross-sectional area of the first flow path is larger than the cross-sectional area of the second flow path.

  The disc-shaped analysis chip of the present invention can more precisely control the profile of multistage liquid feeding by changing the centrifugal force due to rotation.

It is a schematic diagram which shows the outline | summary of the measuring system of this invention. (A) is a figure showing one embodiment of a disk type analysis chip of the present invention. (B) is a perspective view which shows the characteristic part (flow-path part) of the disk type | mold analysis chip shown to (a). It is a top view of the characteristic part (flow-path part) of the disk type | mold analysis chip of this invention. It is a top view which shows another form of the characteristic part (flow-path part) of the disk type | mold analysis chip of this invention. 6 is a graph showing the relationship between the ratio of the liquid fed to the reservoir and the rotation speed in Example 1. It is a graph which shows the relationship between the ratio sent to the reservoir in Example 2, and rotation speed. It is a graph which shows the relationship between the ratio sent to the reservoir | reserver in Example 3, and rotation speed.

  FIG. 1 shows an outline of a measuring apparatus to which the disk type analysis chip of the present invention is applied. The disk type inspection chip 1 is installed on a turntable 21 and can be rotated by a motor 22. Various fluid treatments in the flow path provided in the disk-type analysis chip 1 can be performed by sequentially changing the centrifugal force from the center to the outer peripheral direction with respect to the disk-type analysis chip. The centrifugal force is applied to the disk-type analysis chip by placing and rotating the disk-type analysis chip 1 on a device (centrifuge) that can apply the centrifugal force, such as the motor 22 and the turntable 21. Done. By changing the rotation speed of the turntable 21, the centrifugal force applied to the disk-type analysis chip can be changed.

  The liquid introduced into the following measuring tank in the outer peripheral portion by the centrifugal force due to rotation is irradiated with an electromagnetic wave for excitation from the light source 31, and the fluorescence excited by the fluorescent substance in the liquid is detected by the photodetector 32. As the light source 31, an LED, an LD, or the like can be used, and as the light detector 32, a PD, APD, PM, or the like can be used.

  FIG. 2A shows an embodiment of the disk-type analysis chip of the present invention. On the surface of the disk-type analysis chip 1, a flow path portion from the center toward the outer peripheral portion is provided. FIG. 2B shows details of each flow path portion. As shown in FIG. 2B, a sample tank 11, a first flow path 101, a reaction tank 12, a second flow path 102, and a measurement tank 13 are provided from the center side of the disk-type analysis chip. The reaction tank 12 is filled with beads 4 to which proteins are bound. Here, the cross-sectional area of the first channel 101 is designed to be larger than that of the second channel 102.

  An example of a method for measuring IgA (antigen) or the like in a specimen using the disc-type analysis chip of the present invention will be described with reference to FIG. First, a sample (saliva, blood, etc.) and a reagent (antibodies modified with a fluorescent dye) are injected into the sample tank 11, and the disk-type analysis chip 1 is increased to an appropriate number of revolutions to work in the direction of the arrow in the figure. The specimen is introduced into the reaction tank 13 by centrifugal force. The appropriate number of revolutions is a speed at which the specimen flows through the first channel 101 and moves to the reaction tank 13 but does not flow into the second channel 102. By maintaining the number of rotations for a certain period of time or stopping the rotation, the reaction time between the beads and the specimens and reagents modified with biomolecules (such as the antibody antigens) previously filled in the reaction tank 12 is secured. Thereafter, the disk-type analysis chip 1 is rotated at a higher rotational speed, so that a liquid containing unreacted reagents and the like flows through the second flow path 102 and moves to the measurement tank 13. By measuring the liquid in the measurement tank 13 with the light source 31 and the photodetector 32, the measurement target (such as an antibody) in the sample can be quantified. If a stepping motor is employed as the motor 22, the plurality of measurement tanks 13 arranged on the disk-type analysis chip can be sequentially moved to the position of the photodetector 32 to detect fluorescence.

  The material of the disc-shaped analysis chip is not particularly limited. For example, polymethyl methacrylate resin (PMMA), polydimethylsiloxane (PDMS), glass, cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene terephthalate (PET), polystyrene (PS), and polypropylene (PP). In order to produce industrially, it is preferable to use PMMA, PET, COP, and COC. When the measurement in the measurement tank is measurement of the amount of fluorescence detected, it is preferable that at least the material constituting the measurement tank is a material that hardly excites fluorescence. Examples of such materials include PMMA, COP, and COC.

The thickness of the disc-shaped analysis chip is not particularly limited, but is preferably 0.1 to 100 mm, more preferably 2 to 3 mm. The method of forming the flow path portion on the disk-shaped substrate is not particularly limited, but any width of 1 μm to 10 mm or a width of 1 μm to 10 mm is used on the substrate by using any one of machining, sandblasting, injection molding, or a combination of a plurality of methods. Examples include a method of forming a flow path having a depth and joining a plurality of (usually two) substrates, films and the like by methods such as thermocompression bonding, adhesion, and fitting. The cross-sectional area of the formed flow path is preferably 10 μm ^{2 to} 10 mm ² (the depth is preferably 5 μm to ⁵ mm).

  It is preferable that each flow path formed in the disk-type analysis chip does not communicate with the outside other than connection with each tank. This is because the sample and the reagent may flow out to the outside and a measurement error may occur due to the deterioration of the sample and the reagent.

  The inner surface of each flow path (such as the first flow path and the second flow path) formed in the disk-type analysis chip is preferably formed from a material having a large contact angle (a material having high water repellency). . This is because when the contact angle of the inner surface of the flow path is small (high hydrophilicity), the liquid moves between the tanks due to the capillary phenomenon, which makes it difficult to control liquid feeding. For this purpose, it is preferable to have a water repellent coating layer on the inner surface of each flow path. By having a water-repellent coating layer, all the liquid is fed from the center side tank to the outer side tank (for example, from the sample tank to the reaction tank) with a slight change at a constant rotational speed, and the liquid feed control is performed. This is because it tends to be easy. Moreover, the effect which prevents the adsorption | suction of the foreign material and protein to a flow path can also be anticipated. Note that the same water-repellent coat may be applied to all of the flow paths, or another type of water-repellent coat may be applied to each portion. However, it is desirable not to form a water repellent coating layer on the inner surface of the sample tank. This is because inconvenience may occur when the specimen is injected into the sample tank.

  The material of the water-repellent coating tank is not particularly limited, but a material that does not easily excite fluorescence is preferable, and a material that does not fog or dissolve the surface of the substrate material of the disk-type analysis chip is preferable. Specifically, when an acrylic resin is used as the substrate material of the disk-type analysis chip, for example, Fluorosurf (registered trademark) manufactured by Fluoro Technology, Cytop (registered trademark) manufactured by Asahi Glass Co., Ltd., Adesso manufactured by Nikka Chemical Co., Ltd. (Registered trademark). Of these, a fluorosurf manufactured by Fluoro Technology, Inc. is preferable.

  Note that the disc-shaped analysis chip does not necessarily have a disc shape, and other shapes such as a rectangle can be adopted as long as the disc can be rotated in a balanced manner. The disk-type analysis chip may be configured by installing each divided chip in a disk-type chip holder in which a plurality of divided chips can be installed.

  In the case where the sample tank has an injection port for injecting a sample, a foreign substance may be prevented from being mixed before the sample is injected, and a seal covering the injection port may be attached after the sample is injected. The seal may be written with symbols, numbers, explanatory notes, etc. so that the number of the injection port into which the specimen has been injected corresponds to the output result from the detector.

  The reaction in the reaction vessel is not particularly limited, and examples thereof include an antigen-antibody reaction, a reaction between a protein and a ligand, and a hybridization reaction.

  The reaction vessel is preferably filled with beads for immobilizing proteins, DNA, biomolecules and the like. The beads are not particularly limited, and examples thereof include plastic beads, glass beads, and metal (having magnetic properties) beads. The diameter of the beads is preferably 1 μm to 10 mm, more preferably 0.1 to 1.0 mm, and it is preferable to have a size that does not flow into the second flow path. The shape of the beads is not particularly limited, and examples thereof include spheres and cubes. A plurality of beads may be combined and filled. Further, beads made of a porous material may be used.

  The beads are usually modified with biomolecules necessary for the various reactions described above. For example, when the reaction is an antigen-antibody reaction and an antibody in a specimen is measured, it is preferable to modify the same antibody or antigen on the beads. In this case, an antigen-antibody reaction is performed by injecting a specimen containing an antibody and a reagent containing an antigen-labeled complex into a sample tank and sending the reagent to the reaction tank. Thereafter, an unreacted antigen-labeled complex is included. The liquid is sent to the measuring tank. By irradiating the liquid stored in the measurement tank with an electromagnetic wave that excites fluorescence and measuring the excited fluorescence, the antibody in the specimen can be quantified.

  As shown in FIG. 3, the reaction tank 12 and the measurement tank 13 are preferably provided with exhaust ports 121 and 131. This is because if there is no exhaust port, it is difficult to cause the liquid to flow through the flow path due to atmospheric pressure. At this time, the position where the exhaust ports 121 and 131 are provided is preferably closer to the center side (upstream side) of the disc type analysis chip than the reaction tank 12 and the measurement tank 13. This is to prevent the liquid in the reaction tank 12 and the measurement tank 13 from flowing out from the exhaust ports 121 and 131 due to the centrifugal force acting in the direction of the arrow in the figure. In particular, when air enters the measurement tank 13, the measurement value is affected, so that it is filled with the liquid. For the same reason, the exhaust ports 121 and 131 are preferably connected to the center side of the disc type analysis chip of the reaction tank 12 and the measurement tank 13 by the exhaust pipes 122 and 132, respectively.

  In the above description, the two-stage liquid feeding mechanism has been described. However, the disc type analysis chip of the present invention may have a structure having three or more stages of liquid feeding mechanisms. FIG. 4 shows a flow path portion of an example of the disc type analysis chip of the present invention having a three-stage liquid feeding mechanism. In the disc type analysis chip shown in FIG. 4, a reagent vessel 14 connected to the center side (upstream side) of the disc type analysis chip from the reaction vessel 12 by a third flow path 103 separately from the sample vessel 11. Is equipped. Here, the cross-sectional area (thickness) of each flow path is designed so that the first flow path 101> the third flow path 103> the second flow path 102. By increasing the rotational speed of such a disk-type analysis chip stepwise, three-stage liquid feeding becomes possible. That is, first, the sample in the sample tank 11 is introduced into the reaction tank 12 by increasing the rotation speed to an appropriate speed, and then the reagent in the reagent layer 14 is introduced into the reaction tank 12 by further increasing the rotation speed. By increasing the rotation speed, the reaction liquid of the specimen and the reagent can be introduced into the measurement tank 13.

  The measurement system using the disk-type analysis chip preferably has a function of displaying the measurement result by itself. This is because it is easy to carry and can be used in various situations such as measurement at home. Alternatively, it may be connected to an external control device such as a computer, the measurement result is displayed by a display function on the control device, and control may be performed by software. For example, according to instructions from the computer based on the software, samples and reagents are injected into the disk-type analysis chip, the rotation speed of the disk-type analysis chip is controlled, and graphs of measurement results and measurement values are displayed on the computer screen Can be displayed. In this case, the number of components of the measurement system other than the computer is reduced, and a small and inexpensive system can be provided.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

Example 1
<Preparation of test disc type analysis chip>
An NC milling machine is provided with two disk-shaped plate bodies made of PMMA, one tank, a tank on the outer peripheral side of the disk-shaped plate body with respect to the tank, and a recess for forming a flow path connecting them. A flow path along the radial direction of the two tanks and the disk-shaped plate connecting the two tanks by thermocompression-bonding the surfaces having the concave portions of the two disk-shaped plate-shaped bodies formed by a processing machine. A test disk type analysis chip having 24 sets of sample tank, reaction tank and first flow path, or reaction tank, measurement tank and second flow path) in the disk type analysis chip of the present invention Was made. The size of the tank on the center side is 3 mm in diameter and 1 mm in depth, and the size of the tank on the outer peripheral side is 3 mm in diameter and 1 mm in depth. The length of the flow path is 25 mm. An exhaust port was formed in the outer peripheral side tank by an NC milling machine.

  In this example, six types of chips with different flow path depths were produced. As a result of measuring the depth of the channel of each chip using a laser confocal microscope (manufactured by Lasertec Corporation), they were 39, 50, 84, 90, and 117 μm.

<Liquid feeding test>
Colored water was injected in an amount of 5 μl into the central tank of the test disc type analysis chip produced above, and the disc type analysis chip was placed on a turntable and first rotated at 100 RPM. Thereafter, while increasing the rotational speed by 100 rpm, maintaining for 30 seconds at each rotational speed was repeated to finally increase the rotational speed to 3000 rpm. During that time, the position of the liquid that progressed toward the outer peripheral side reservoir (reservoir) was sequentially measured by visual observation. A graph summarizing the measurement results is shown in FIG.

  From the results shown in FIG. 5, it can be seen that in the case of a chip having a flow path depth of 84 to 117 μm, all the liquid is sent to the reservoir when a certain number of rotations is exceeded. However, when the depth of the flow path is 50 μm, liquid feeding to the reservoir is performed in stages, and when the depth of the flow path is 39 μm, liquid feeding is not performed at all even if the rotational speed is increased to 3000 rpm. I wasn't.

(Example 2)
<Preparation of test disc type analysis chip>
Except that after forming a recess in two disk-shaped plates, the surface of the recess was coated with Fluorosurf (registered trademark) FS-1060TH-0.5 (manufactured by Fluoro Technology) as a water-repellent coating layer. Produced a test disk type analysis chip in the same manner as in Example 1. In this example as well, six types of chips with different flow path depths were produced. As a result of measuring the depth of the flow path in the same manner as in Example 1, they were 37, 50, 90, 109, 113, and 130 μm.

<Liquid feeding test>
About the test disc type | mold analytical chip produced above, it carried out similarly to Example 1, and measured the liquid amount sent to the tank (reservoir) of the outer peripheral part side. A graph summarizing the measurement results is shown in FIG.

  From the results shown in FIG. 6, it can be seen that in all the chips having a flow path depth of 50 to 130 μm, all the liquid is sent to the reservoir when a certain rotation speed is exceeded. In addition, when the depth of a flow path is 37 micrometers, although a liquid feeding is performed in steps, it can be used for the measuring system of this invention by enlarging the raise range of rotation speed. From this result, it can be seen that by applying a specific water-repellent coating on the surface, liquid feeding can be controlled even with a narrower flow path by a normal centrifugal device of 3000 rpm or less.

(Example 3)
<Preparation of test disc type analysis chip>
After forming the recesses in the two disk-shaped plates, the surface of the recesses was coated with Fluorosurf (registered trademark) FS-1060TH-0.5 (manufactured by Fluoro Technology Co., Ltd.) as a water-repellent coating layer and discharged by air blow A test disc type analysis chip was prepared in the same manner as in Example 1 except that the coating was dried and coated. In this example as well, six types of chips with different flow path depths were produced. The depth of the channel was measured in the same manner as in Example 1. As a result, it was 40, 65, 79, 110, 120, and 159 μm.

<Liquid feeding test>
About the test disc type | mold analytical chip produced above, it carried out similarly to Example 1, and measured the liquid amount sent to the tank (reservoir) of the outer peripheral part side. A graph summarizing the measurement results is shown in FIG.

  From the results shown in FIG. 7, it can be seen that in all the chips having a flow path depth of 40 to 159 μm, all the liquid is sent to the reservoir when a certain number of rotations is exceeded. In particular, even in the case of a thin channel having a channel depth of 40 μm, there was no gradual liquid feeding tendency, and all of the liquid was fed to the reservoir as the number of rotations increased by one step. From this result, it can be seen that by applying a specific water-repellent coating on the surface, liquid feeding can be controlled even with a narrower flow path by a normal centrifugal device of 3000 rpm or less.

  DESCRIPTION OF SYMBOLS 1 Disc type | mold analysis chip, 101 1st flow path, 102 2nd flow path, 103 3rd flow path, 11 Sample tank, 12 Reaction tank, 121,131 Exhaust port, 122,132 Exhaust pipe, 13 Measurement tank 14 reagent tanks, 21 turntables, 22 motors, 31 light sources, 32 photodetectors.

Claims (6)

A disk-type analysis chip is placed on a centrifuge and used for a measurement system that performs measurement after moving the sample on the disk-type analysis chip using the centrifugal force generated by the rotation of the centrifuge and reacting with the reagent. A disc-shaped analysis chip,
On the surface of the disc type analysis chip, a sample tank, a reaction tank provided in the direction of the outer periphery of the disc type analysis chip with respect to the sample tank, and provided in the direction of the outer periphery of the disc type chip with respect to the reaction tank A measurement tank, a first flow path connecting the sample introduction tank and the reaction tank, and a second flow path connecting the reaction tank and the measurement tank,
The disk-type analysis chip, wherein a cross-sectional area of the first flow path is larger than a cross-sectional area of the second flow path.   The disk-type analysis chip according to claim 1, further comprising a water-repellent coating layer on an inner surface of the first flow path and the second flow path.   The disc-shaped analysis chip according to claim 1 or 2, wherein the measurement is measurement of a fluorescence detection amount, and a material constituting the measurement tank is a material that hardly generates fluorescence.   The disk-shaped analysis chip according to claim 3, wherein the material that hardly generates fluorescence is a (meth) acrylic resin or a cycloolefin resin.   The disk-type analysis chip according to any one of claims 1 to 4, wherein the reaction tank and the measurement tank have an exhaust port. A measurement system including a centrifuge device and a disk-type analysis chip installed on the centrifuge device,
Using the centrifugal force generated by the rotation of the centrifuge, the sample on the disk-type analysis chip is moved and measured after reacting with the reagent.
On the surface of the disk-shaped chip, a sample tank, a reaction tank provided in the direction of the outer periphery of the disk-shaped chip with respect to the sample tank, and a measurement tank provided in the direction of the outer periphery of the disk-shaped chip with respect to the reaction tank A first flow path for connecting the sample introduction tank and the reaction tank, and a second flow path for connecting the reaction tank and the measurement tank,
The measurement system, wherein a cross-sectional area of the first flow path is larger than a cross-sectional area of the second flow path.

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