KR20130030665A - Rotor assembly of cetrifugal separator for automatic extraction of biological material and extraction method of biological material using the same - Google Patents

Abstract

PURPOSE: A rotor assembly of a centrifugal separator for the automatic extraction of biological materials is provided to easily, quickly, and accurately extract biological materials and to prevent noise. CONSTITUTION: A rotor assembly of a centrifugal separator for the automatic extraction of biological materials comprises a rotor(10) which has a partition(14) dividing the inner space into a plurality of divided spaces and a rotary shaft insertion hole(13) formed in the center of the inner space; a plurality of racks(21) which are removably accommodated in the divided spaces and form a plurality of tube insertion spaces; and a plurality of tubes which are removably inserted in the tube insertion spaces; and a front charging tube which is removably inserted in the upper part of the tubes.

Description

Rotor assembly of cetrifugal separator for automatic extraction of biological material and extraction method of biological material using the same}

The present invention relates to a centrifugal separator for automatic extraction of biological material and a method for extracting biological material using the same. More particularly, the present invention relates to a rotor assembly, a reagent tube, and a method for extracting biological materials using the same, which are provided in a centrifugal separator capable of automatically extracting a specific material from a sample of biological material.

Today, the bio industry is the fastest growing sector among the high-tech industries that will determine national competitiveness in the 21st century. Molecular diagnostic technology using genes is expected to grow at an annual average rate of 13%. The process for molecular diagnosis is through gene extraction, gene amplification, and gene analysis. Extracting genes such as DNA and RNA from biological materials has been commercialized with appropriate extraction kits and buffers according to the target genetic material.In general laboratories, centrifuge and pipette work using commercially available extraction kits and solutions. By using the operator, the sample has been manually separated and extracted. On the other hand, in some public institutions and large health examination centers, skilled workers extract biological materials using expensive automatic extraction systems.

Various methods have been developed for separating nucleic acids and proteins from biological samples. For example, precipitation, liquid extraction, electrophoresis, chromatography, and the like have traditionally been used, and solid phase extraction has been developed to simplify such operations. This solid phase extraction method uses solid particles having a selectivity or solid particles prepared by attaching a ligand having a high selectivity to a solid phase. Solid phase extraction method has been used to fill the column with solid particles or filter membrane to the column.

In this case, a filter-type membrane is used when there are few fine particles or samples having a large surface area to increase the adhesion capacity. However, there is a problem in that the solution slowly flows between the fine pores when filled with such fine particles or using a membrane in the form of a filter. Therefore, in order to flow the solution rapidly, a centrifugal separator is used to increase the gravity value or apply a pressure difference by applying a vacuum. Alternatively, by using fine magnetic particles with a large surface area, biochemicals can be quickly attached in the suspension state of a solution, magnetic fields can be agglomerated by applying a magnetic field, and the solution can be removed. Technology has been developed since the ages.

In general, the centrifugation process for extracting genes of a biomaterial is performed by attaching the genes to the filter column tube (hereinafter referred to as the filter tube) through two or three washing processes, separating the rest outside the washing tube, and then applying the genes to the filter. The tube is transferred to an elution tube and subjected to an elution process using a reagent buffer such as ethanol. In other words, binding process for attaching biological material (blood, cell, tissue, etc.) containing gene to filter of tube, washing process for attaching gene only to filter and filtering residue Elution process to extract only the gene (DNA, RNA) will be included.

Most laboratories use centrifuges and tubes with filters and extracting reagents for these biomaterial separation and extraction devices. It is an inefficient work structure in which excessive labor is consumed. Differences in reproducibility and uniformity occur depending on the skill of the operator, and there has always been a concern about DNA / RNA purity and cross contamination. In addition, since the work must be performed according to a predetermined extraction protocol, mistakes in the extraction process by hand are occurring at random.

In some government laboratories and large health examination centers that require large amounts of sample processing to overcome this problem, automatic extraction equipment is used for productivity and accuracy, but the equipment is expensive. It is not easy to expand the scope to many customers such as general laboratories, intermediate hospitals, bio companies, and public health centers. Genetic extraction equipment has been in need of localization by importing over 90% of expensive automatic equipment.

On the other hand, the current gene extraction method can be classified into two types. By using the principle of magnetic, DNA and RNA can be distinguished into magnetic bead method that attaches and extracts magnetic beads to the magnetic beads and membrane filter method that binds DNA and RNA to the membrane filter.

Magnetic bead type automatic extraction equipment is easy to configure. This method has a large number of simultaneous extractions and a variety of prices. The magnetic bead method is capable of mass extraction, and it is convenient for use, but it is difficult to completely dry the reagent from the beads during elution, which is the final step of the extraction process due to the high cost of extracting beads and reagents. have.

Membrane filter method is divided into centrifugal separation method and extraction method of applying air to the column using vacuum pressure. Double centrifugation is superior to the method using vacuum pressure. The centrifugal separation method is better in terms of extraction purity and performance, and it is inexpensive per extraction because it uses filter tube which is generally used for manual labor in a general laboratory and is familiar to general experimenters. However, this method requires high-speed rotation, and the automatic extraction requires a moving process of the filter tube and a mechanism for automatic dispensing of the extraction reagent, which makes it difficult to automate, causing problems such as cross contamination and cost increase.

It is an object of the present invention to provide a rotor assembly of a centrifugal separation device for extracting biological material, an extraction reagent tube structure, a reagent injection method, and a principle separation method using the same, which can solve the problems of the prior art.

Another object of the present invention is to provide a rotor assembly of a centrifugal separation device for automatic extraction of biological material which can be easily and quickly and accurately carried out, and a principle separation method using the same.

Another object of the present invention is to prevent contamination in the process of injecting the reagent into the tube, the noise can be prevented during centrifugation, the injection of reagents and the replacement of the tube can be performed automatically of the biological material It is to provide a rotor assembly of a centrifugal separator for automatic extraction and a principle separation method using the same.

In order to solve the above object, according to the present invention, a rotor having a partition wall for dividing the inner space into a plurality of divided spaces and a rotation shaft insertion hole formed in the center of the inner space; A plurality of racks removably received within the divided spaces of the rotor, the plurality of racks being inclined upon rotation of the rotor and forming a plurality of tube insertion spaces; A plurality of tubes, which can be removably inserted into a plurality of tube insertion spaces provided in the plurality of racks; And, a prefilled tube that can be removably fitted on top of the plurality of tubes. A rotor assembly of a centrifugal separator for automatic extraction of biological material is provided.

According to one feature of the invention, the plurality of tube insertion spaces are formed in a tube holder that can be removably received in each of the plurality of racks.

According to another feature of the invention, the swing shaft protrudes from the partition wall, the swing shaft is coupled to the shaft insertion groove formed in the side wall of the rack, so that the rack rotates about the swing axis when the rotor assembly is rotated Tilted.

According to another feature of the invention, at least one of the plurality of tubes, the filter is provided inside the tube and the bottom is open.

According to another feature of the invention, the precharge tube has a body portion having one or more isolation spaces formed thereon, and an extension portion extending from the body portion and having an opening formed at the bottom thereof, each of the one or more isolation spaces having a bottom portion. By drilling the bottom of the corresponding body part, the solution filled in the respective isolation space can be discharged through the opening of the extension part.

According to another feature of the invention, there is further provided a bearing for rotatably supporting the upper end of the rotor and a support member provided with the bearing.

According to another feature of the invention, the precharge tube is a body portion formed with at least one isolation space, an extension portion extending from the body portion and the opening is formed in the bottom, coupling for forming on the outer surface opposite to each other the body portion And a protrusion and a coupling recess, wherein the coupling protrusion or coupling recess of one precharge tube may be coupled or separated from each other with the coupling recess or coupling protrusion of the other precharge tube. By drilling the bottom of the body portion corresponding to each bottom of the above isolation space, the solution filled in each isolation space can be discharged through the opening of the extension portion.

According to another feature of the invention, in order to measure the amount of rotation of the rotor assembly, a light receiving sensor capable of receiving light of a light emitting portion installed in the camera or the rotor assembly capable of imaging the marking (marking) displayed on the rotor assembly is further Include.

According to another feature of the present invention, the rotor further includes a chamber disposed outside the rotor to accommodate the inside, and a fan or cooling device capable of blowing cooling air into the chamber.

Further, according to the present invention, in the rotor assembly having a rack installed in a plurality of divided spaces formed by the partition wall, the rack is installed so that the rack can be tilted by the centrifugal force generated when the rotor assembly is rotated, the tube provided in the rack Insert a first to third tube into an insertion space or a tube holder housed in the rack, provide a filter in the second tube, form an opening in the bottom, and insert a prefilled tube into the top of the second tube. Extracting a biological material from each of the first to third containment spaces formed by filling respective reagents or placing a prefilled prefilled tube in a tube holder; In order to separate DNA by adding a pretreatment reagent to a sample of biological material, cells of membranes of cells or tissues are removed, cell debris is removed, or a sample is placed in a first tube and centrifuged according to the sample type. Pretreatment of the biological sample to separate the upper and lower layers; Injecting some or all of the pretreated sample into the second tube and inserting the precharge tube into an upper end of the second tube; Binding to attach a biological dielectric material to a filter provided in a second tube by performing centrifugation using the rotor assembly; Performing a centrifugal separation using the rotor assembly while injecting reagents in the first containment space into the second tube by puncturing the first containment space of the prefilled tube; A second cleaning step of performing centrifugation using the rotor assembly while injecting reagent in the second isolation space into the second tube by puncturing the second isolation space of the precharge tube; Inserting the second tube with the prefilled tube inserted into the third tube and puncturing the third containment space, the reagent in the third containment space passes through the opening at the bottom of the second tube to the third tube. There is provided an extraction method using the rotor assembly of the centrifugal separator for automatic extraction of biological material, comprising; performing centrifugation using the rotor assembly in a flowing state.

According to another feature of the invention, in the rotor assembly having a rack installed in a plurality of divided spaces formed by the partition wall, the rack is installed so that the rack can be tilted by the centrifugal force generated during the rotation of the rotor assembly, provided in the rack A precharge that can insert first to third tubes into an integrated tube insertion space or a tube holder housed in the rack, provide a filter in the second tube, form an opening in the bottom, and fit into an upper end of the second tube Extracting a biological material from each of the first to fourth containment spaces formed in the tube, or placing the prefilled tube in the tube holder; Cell lysis of cells or tissue membranes by adding pretreatment reagents to a sample of biological material, removal of cell debris, or separation of the upper and lower layers by placing the sample in a first tube and centrifuging according to the sample type For pretreatment of the biological sample; Injecting some or all of the pretreated sample into the second tube and inserting the precharge tube into an upper end of the second tube; A binding step of injecting a binding reagent contained in the first isolation space into the second tube by puncturing the first isolation space of the precharge tube, and then attaching the dielectric material to the filter of the second tube while rotating the rotor assembly; Performing a centrifugal separation using the rotor assembly while injecting reagents in the second containment space into the second tube by puncturing the second containment space of the prefilled tube; Performing a centrifugal separation using the rotor assembly while injecting the reagent of the third isolation space into the second tube by puncturing the third isolation space of the precharge tube; Inserting the second tube with the prefilled tube inserted into the third tube and puncturing the fourth containment space, the reagent in the fourth containment space passes through the opening at the bottom of the second tube to the third tube. And performing centrifugation using the rotor assembly in a flowing state.

The rotor assembly of the centrifugal separator for the automatic extraction of biological material and the centrifugal device using the same make it possible to extract the biological material easily and accurately. It also allows to overcome various problems that have been a problem in the prior art. For example, the cleaning reagent and the eluting reagent are precharged in the prefilled tube, the prefilled tube is perforated, and the respective reagents are distributed to the filter tube with the silica membrane filter, thereby making it more remarkable than a single injection amount in the conventional invention. It is possible to prevent cross contamination which can occur during dispensing into the filter tube by suctioning the reagent as much as one injection amount from a container containing a lot of reagents, and it is possible to reduce the cost by not needing a separate mechanism for automatic dispensing. And the size of the appearance can be reduced.

1 is a schematic perspective view of a rotor assembly provided in the centrifugal separator according to the present invention.
FIG. 2 is a schematic perspective view of the rack and tube holder of the rotor assembly shown in FIG. 1. FIG.
3 is a schematic perspective view of the tube holder shown in FIG. 1.
4 is a schematic perspective view of another example of a tube holder.
5 is a cross-sectional view showing a state in which a tube and a precharge tube are inserted into a tube holder.
6 is a schematic perspective view of the precharge tube.
7 is a schematic side cross-sectional view of the rotor assembly shown in FIG. 1.
8a and 8b show schematic side and top views of an automatic extraction device incorporating a centrifugal separator according to the invention.
9A and 9B are schematic perspective views of another embodiment of a precharge tube according to the invention.
10 is a schematic perspective view of another embodiment of a precharge tube according to the invention.
FIG. 11 is a perspective view showing that the joinable prefilled tube shown in FIGS. 9A and 9B is inserted into the tube holder.
12 is a first example of a schematic flowchart for performing extraction of biological material using the centrifugal separator according to the present invention.
13 is a second example of a schematic flowchart for performing extraction of biological material using the centrifugal separator according to the present invention.
14 is a third example of a schematic flowchart for performing extraction of biological material using the centrifugal separator according to the present invention.

1 is a schematic perspective view of the rotor assembly of the centrifugal separator provided in the automatic biological material extraction apparatus according to the present invention.

Referring to the drawings, the rotor assembly 1 includes a rotor 10, a rack 21 accommodated in the rotor 10, and a tube holder 31 fitted to the rack 21. In addition, the rotor assembly 1 may include first to third tubes 51, 52, and 53 inserted into the tube holder 31 and a precharge tube 61 as illustrated in FIG. 5.

The rotor 10 shown in FIG. 1 is a swing-out rotor, which includes a circumferential wall 11 having a circular shape, a partition 14 partitioning a circular space surrounded by the circumferential wall 11, and the rotor ( 10 is provided with a rotating shaft insertion hole 13 is inserted into the rotating shaft for rotating. A rotating shaft (not shown) may be inserted through the rotating shaft insertion hole 13, and the rotating shaft (not shown) may be rotated by a driving motor (not shown). Thus, the rotor 10 can be rotated.

The circular space surrounded by the circumferential wall 11 is divided into a plurality of partitions 12 by the partition wall 14. A rack 21 may be accommodated in the partition 12. In the example shown in the figure, the rotor 10 has four compartments 12. The rack 21 is held rotatably by a swing axis (not shown) projecting from the partition 14 forming the partition 12. That is, the swing shaft protruding from the partition 14 is inserted into the swing shaft insertion grooves (FIGS. 3 and 27) formed in the rack 21, and the rack 21 may be rotated about the swing shaft (not shown). Therefore, when the rotor 10 rotates at a high speed, the rack 21 is rotated so as to be inclined with respect to the vertical axis by the action of the centrifugal force. That is, as shown in FIG. 1, the bottom of the rack 21 faces the radially outer side of the rotor 10, and the top of the rack 21 is inclined toward the radially inner side of the rotor 10.

The rack 21 houses the tube holder 31. The tube holder 31 is for receiving a tube, as will be described in more detail later, wherein one or more tube holders 31 may be received in the rack 21. In the example shown in FIG. 1, four tube holders 31 are accommodated in each rack 21. In another embodiment, not shown in the figure, a swing-out rotor without the circumferential wall 11 may be considered, thus reducing weight.

2 and 3 are perspective views showing the rack and tube holder shown in FIG. 1 in more detail.

Referring to the drawings, the tube holder 31 is accommodated in the rack 21. In the example shown in FIG. 2, only one tube holder 31 is accommodated in the rack 21 and no tube is inserted into the tube holder 31. The tube (not shown) may be inserted into the tube insert 32 formed in the tube holder 31 or removed from the tube insert 32. The rack 21 has a rectangular space consisting of the front wall 22, the back wall 23 and the side walls 24a and 24b. Guide grooves 25 are formed on the inner surfaces of the front wall 22 and the rear wall 23. According to the shape of the tube holder, guide grooves may be formed on the inner side surfaces of the side walls 24a and 24b. The guide groove 25 is formed so that the insertion portion 34 (FIG. 4) formed in the tube holder 31 can be inserted. When the tube holder 31 is accommodated in the rack 21, the guide groove 25 It serves to guide the location. As can be seen from the figure, the front wall 22 is formed at a lower height than the rear wall 23.

The rack shown in FIG. 2 is in a state in which a part of the middle portion of the rear wall 23 is removed (part designated by reference numeral 23a), whereas the rack shown in FIG. 3 is not removed from the wall of the rear wall 23 '. The guide groove 25 'is formed in the vertical direction over the entire inner surface of the rear wall 23'.

As can be seen from FIG. 3, the side wall 24b is formed in the shaft insertion groove 27. The lower end of the shaft insertion groove 27 is formed to be open at the bottom of the side wall 24b, while the upper end of the shaft insertion groove 27 is closed at a position above the middle portion of the side wall 24b. Thus, when the rack 21 is installed in the partition 12 shown in FIG. 1, the swing shaft 72 (FIG. 7) protruding from the partition 14 is inserted from the lower end of the shaft insertion groove 27 and then. After moving upward along the shaft insertion groove 27, the shaft is caught at the upper end of the insertion groove 27. As a result, the rack 21 is in the state which hangs on the swing shaft 72 (FIG. 7). Accordingly, as shown in FIG. 1, when the centrifugal force is applied while the rotor 1 rotates, the rack 21 is rotated and inclined about the swing shaft 72 (FIG. 7).

Shown in FIG. 4 is a schematic perspective view of the tube holder 31. The tube holder 31 is formed with one or more tube inserts 32 as shown in the drawing, and in the example shown in the drawing, three tube inserts 32 are formed. The tube insert 32 is inserted with tubes which will be described in more detail later. An insertion part 34 may be formed at one side of the tube holder 31, and the insertion part 34 may be inserted into the guide grooves 25 and 25 ′ shown in FIGS. 2 and 3 as described above. Can be. In addition, the step portion 33 formed in the tube holder 31 is held on the front wall 22 of the rack 21 shown in Figs. Thus, the tube holder 31 is held in a state of being held by the rack 21, and the tube holder 31 is prevented from escaping through the open bottom of the rack 21.

In another embodiment according to the invention, which is not shown in the drawings, a tube insertion space may be provided in the rack. For example, the rack 21 as shown in FIG. 2 and the four tube holders 31 accommodated in the rack 21 as one member are provided, and the tube insertion opening 32 is provided as a tube insertion space. can do. That is, the tube holder 31 is formed in the rack 21 as an integral member, not a separate member that can be separated from the rack 21. In such a case, there is an advantage that the manufacturing cost is low and the handling is easy. One of ordinary skill in the art, based on the description above and shown in FIG. 2, can implement other embodiments in which a tube insertion space is provided in the rack, examples of which are included within the scope of the present invention.

5 is a schematic cross-sectional view showing a state in which a tube is inserted into the tube holder shown in FIG.

Referring to the drawings, first to third tubes 51, 52, and 53 may be inserted into each tube insertion hole 32 (FIG. 4) of the tube holder 31. The first to third tubes 51, 52, 53 may have different shapes as shown in the figures, or may have the same shape in other embodiments not shown in the figures. The first to third tubes 51, 52, and 53 may be inserted into the tube insertion holes 32 (FIG. 4), and the pre-filled tube 61 may be inserted into the second tube 52. . The prefilled tube 61 is filled with a reagent in advance, and reagents necessary for the action performed in the second tube 52 can be supplied to each of the tubes 51, 52, and 53. The precharge tube 61 may be fitted to or removed from the upper end of the second tube 52 according to a process described in more detail later. In the example shown in the figure, the precharge tube 61 is shown fitted to the upper end of the second tube 52.

The first tube 51 is, for example, a tube for pretreatment for performing centrifugation according to the type of sample. The pretreatment tube receives a pretreated sample as a process before centrifugation, and centrifuged at about 10,000 rpm or more. After centrifugation, the sample appears to be separated into the supernatant and the lower layer in the pretreatment tube, where only the supernatant is taken and transferred to the second tube 52. In other cases, if the nucleic acid is to be extracted from blood, pretreatment tubes may not be used.

The second tube 52 is for example a filter tube. The filter tube is provided with the filter 55 in the lower part, and the opening 52a is formed in the lower part.

The filter tube, which is the second tube 52, comprises a lower portion having a relatively small diameter and an upper portion having a relatively large diameter, so that a step is formed in the tube. The step formed in the second tube 52 is caught by the insertion step step portion 32a formed in the tube holder 31, so that the second tube 52 no longer moves toward the bottom surface of the tube holder 31. The lower diameter of the second tube 52 allows the second tube 52 to be inserted into the third tube 53 which will be described later.

The sample injected into the filter tube corresponding to the second tube 52 is centrifuged by rotating at 10,000 rpm or more while the precharge tube 61 is fitted at the top. As will be described in more detail below, before the centrifugation using a filter tube is performed, a hole is drilled in a portion of the precharge tube 61 having a plurality of isolation spaces, whereby the precharge tube ( From any of the spaces of 61) binding reagents, cleaning reagents can be injected into the filter tubes according to the extraction process. While centrifugation with the second tube 52 is performed, the object (e.g., gene) to be extracted from the sample is attached to the filter 55, while the remainder not attached to the filter 55 is removed. 2 it exits to the lower part through the opening 52a formed in the lower part of the tube 52.

The third tube 53 is, for example, an elution tube. In order to perform centrifugation using an elution tube, a second tube 52 may be inserted into the third tube 53. As described above, the second tube 52 is stepped in the middle by different diameters of the upper and lower portions, a second tube 52 having a small diameter is inserted into the third tube 53, and Steps formed in the two tubes 52 are caught on the upper end of the third tube 53. During the centrifugation using the third tube 53, not only the second tube 52 is inserted into the third tube 53, but also the precharge tube 61 is placed on top of the second tube 52. Keep inserted. At this time, by drilling another one of the plurality of sequestration spaces formed in the precharge tube 62, an object (e.g., a gene to which the eluting reagent from the sequestration space is attached to the filter 55 of the second tube 52). To elute). During centrifugation, the eluted objects and reagents are collected into the third tube 53 through the opening 52a formed at the bottom of the second tube 52. The above description with respect to the first to third tubes is exemplary and the present invention is not limited to the above description.

6 is a perspective view illustrating the precharge tube 61 shown in FIG. 5 in more detail.

Referring to the drawings, the precharge tube 61 has an upper body portion 68 and the lower portion of the extension (69). The upper body portion 68 has a plurality of isolation spaces separated by the partition wall 62, and three isolation spaces 63, 64, and 65 are formed in the example shown in the figure. The lower extension 69 is hollow cylindrical and has an opening 66 formed therein. The bottom face of the main body 68 is sealed. The reagent filled in the isolation spaces 63, 64 and 65 perforates the bottom surface of the body portion 68 corresponding to the lower portion of each of the isolation spaces 63, 64 and 65, thereby providing a hollow internal space of the extension 69. Can flow. Perforations at the bottom of the body 68 are made separately for each isolation space 63, 64, 65. Reagents of the prefilled tube 61 flowing through the perforated hole can then be introduced into the second tube 52 through the opening 66. Bottom drilling of body portion 68 may be through a needle or other drilling means.

The sequestration spaces 63, 64, 65 of the precharge tube 61 may be prefilled with different kinds of reagents, and the upper end of the precharge tube 61 may be sealed with a sealing tape, for example. For example, the first isolation chamber 63 is filled with a first cleaning reagent, the second isolation space 64 is filled with a second cleaning reagent, and the third isolation space 65 is eluted. Reagent) can be charged.

In another example, not shown in the figure, the number of isolation spaces formed in the precharge tube can be increased, and the type of reagent prepared can be increased. In another example, the precharge tube may be distinguished from the first precharge tube filled with only the cleaning reagent and the second precharge tube filled with the eluting reagent. In addition, various types of precharge tubes may be mounted on the tube holder 85 at the top of the table shown in FIG. 8, and the precharge tubes suitable for different kinds of extraction objects or extraction procedures may be selectively used. In another example, not shown in the figure, four sequestration spaces may be formed in the prefilled tube, and at least one of the four isolation spaces may be filled with a binding reagent. The binding reagent serves to allow the dielectric material to attach to the membrane filter.

7 is a side cross-sectional view illustrating a state in which a rack, a tube holder, a tube, and the like are assembled to the rotor shown in FIG. 1. Shown on the left side of the drawing is a state in which the rotor is not rotated, while the rack 21 is maintained in a vertical state, while the right side is shown in a state where the rotor 10 is rotating. As the rack 21 is rotated about the swing shaft 72 by the centrifugal force, the rack 21 is inclined. Although not shown in the drawings, a lid suitable for the circumferential wall 11 may be covered on the upper portion of the rotor 10. The circumferential wall of the portion of the rotor circumferential wall where the rack rotates may be removed to reduce the weight of the rotor. Soundproof material may be attached to the bottom of the lid (not shown). The lid and sound insulation attached to the lid serve to suppress noise generated during the centrifugal action. In addition, the upper end of the rotating shaft to support the bearing 75 and the support member 77 in order to prevent the vibration during the rotation of the rotor can serve to reduce the vibration during high speed rotation. The support member 77 has a bar shape (in the form of a narrow member) provided to cross the upper end of the chamber 81a, and a bearing 75 is installed on the support member 77 to form a rotating shaft. Support 71. Since the rotation shaft 71 is inserted into the rotation shaft insertion hole 13 (FIG. 1) formed at the center of the rotor 10, the rotor 10 may be rotated by the rotation driving of the rotation shaft 71.

8A and 8B show schematic side and top views of an automatic extraction device of a biological material according to the present invention.

Referring to FIG. 8A, an X-Y Cartesian robot 82 is provided on an upper portion of the table 81, and a rotor 10 and a motor 86 of a centrifugal separator are provided on one side of the table 81. By rotating the rotor 10 by the driving force of the motor 86 can be a centrifugal action. The robot 82 may be provided with respective means for performing a predetermined action, such as a gripper, a pipette, a suction and dispensing pump, a puncture needle, and the like, as known in the art, and they are lifted and lowered as needed. By the action of the part 83, it can go up and down. The gripper provided in the robot 82 can grip the tube and precharge tube described above and insert it into or remove it from the tube insert 32 of the tube holder 31. In addition, the pipette 84 and the suction and vacuum pump (not shown) may be used to inhale and inject the supernatant of the first tube into the second tube or to dispense and fill the reagent into the prefilled tube. The puncturing needle may puncture the bottom of the precharge tube 61. Used pipettes or puncture needles can be automatically replaced or cleaned and reused after a single use to prevent contamination or after a specified number of uses to prevent contamination.

Meanwhile, the space formed between the rotor 10 and the table 81 in FIG. 8A is a chamber 81a for accommodating the rotor 10. As the rotor rotates, the air temperature in the chamber 81a increases, so that a cooling device is preferably installed. The cooling apparatus may be implemented using a fan (not shown), and the fan may blow air to allow the air to be discharged from the lower end of the chamber 81a to the outside through the centrifuge lid. In order to increase the cooling efficiency, a cooling gas may be used or air by a cooling fan may pass through the Peltier element and be introduced into the chamber as cooler air. In addition, a cooling device for cooling the centrifuge lid may be installed, or the lid may be configured in a structure that can transfer heat well to the outside.

Referring to FIG. 8B, it can be understood that the robot 82 performs an XY Cartesian coordinate movement in the upper plane of the table 81, and the robot 82 moves close to the rotor 10 of the centrifugal separator so that the tube It can be understood that the action of gripping or inserting the precharge tube into the upper end of the second tube can be performed. On the other hand, the upper surface of the table 81 is provided with a precharge tube holder 85, so that the gripper or pipette by the robot 82 between the tubes and the rotor 10 mounted on the precharge tube holder 85. It is possible to perform a predetermined action depending on the program while reciprocating.

The rotor assembly by a rotating body such as a motor maintains a rotation angle from a predetermined position after rotation. In order to measure the rotation angle from the specific position, a system for measuring the amount of rotation can be constructed by using a camera or by using a sensor. For example, the amount of rotation can be measured by displaying a marking at any position in which the rotor assembly 1 rotates, and imaging the marking with a camera fixedly unrotated. In another example, the light emitting unit is installed at an arbitrary rotational position of the rotor assembly 1, and a light receiving sensor capable of receiving light generated by the light emitting unit is installed at a fixed position that is not rotated to count light received by the light receiving sensor. The amount of rotation can be measured by doing this.

9A and 9B are perspective views of another embodiment of the precharge tube according to the present invention.

Referring to the drawings, the joinable prefilling tube 90 is for coupling formed on the side of the main body 99, an extension 69a extending below the main body 99, and the main body 99. The protrusion part 95 and the coupling recessed part 96 are provided, and isolation spaces 63a, 64a, and 65a are formed inside the main body part 99. The isolation spaces 63a, 64a, and 65a are formed in the main body 99 and the extension 69a is formed below the main body 99. The main body of the precharge tube described with reference to FIG. The isolation spaces 63, 64, 65 are formed in 68 and the extension 69 is formed under the main body 68. Although not shown in FIG. 9A, a hole is formed in the bottom of the extension 69a, and a hole is formed through the hole formed in the bottom of the extension 69a by drilling the bottom of each isolation space 63a, 64a, 65a. It is also the same as the configuration described with reference to the precharge tube shown in FIG.

The main body portion 99 shown in FIG. 9A has a rectangular cross section and has a first face 91, a second face 92, a third face 93 and a fourth face 94. As shown in the figure, the first face 91 and the second face 92 are opposite each other, and the third face 93 and the fourth face 94 are opposite each other. A coupling protrusion 95 is formed on the first surface 91, while a coupling recess (not shown) is formed on the second surface 92. A coupling protrusion (not shown) is formed on the third face 93, while a coupling recess 96 is formed on the fourth face 94. Thus, by inserting the joining protrusion 95 of any prefilled tube 90 into the joining recess of another prefilled tube, a plurality of joinable prefilled tubes can be joined to each other. Referring to FIG. 9B, a plurality of joinable prefilled tubes are shown coupled to one another.

10 shows a schematic perspective view of an integrated precharge tube.

Referring to the drawings, the integrated precharge tube has an integral body portion 68b and a plurality of extensions 69b extending from the integral body portion 68b, and an isolation space 63b inside the integral body portion 68b. 64b and 65b are formed. In the example shown in the figure, four extension portions 69b are formed, and isolation spaces 63b, 64b, and 65b are formed at positions corresponding to the respective extension portions 69b. As described with reference to FIG. 6, the reagent injected into the isolation space flows to the extension 69b by drilling the bottoms of the isolation spaces 63b, 64b and 65b, and through the holes formed in the extension 69b. May spill.

11 is a perspective view showing that the joinable prefilled tube is applied to the tube holder.

Referring to the drawings, the rack 22 accommodates the tube holder 31. The rack 22 and holder 31 are the same as described with reference to FIGS. 2 to 5. That is, in the example illustrated in FIG. 2, only one tube holder 31 is accommodated in the rack 22, but in the example illustrated in FIG. 11, four tube holders 31 are accommodated in the rack 22. It is.

The joinable prefill tube 90 is shown in a state where the four are coupled to each other. An extension 69a of the engageable prefill tube 90 which is in the engaged state may be inserted into the second tube 52 of each of the tube holders 31. That is, in inserting the precharge tube into the second tube 52 as described with reference to FIG. 5, the extension part can be simultaneously extended to four second tubes 52 using the joinable precharge tube 90. 69a can be inserted. This simultaneous insertion action enables more convenient and quick operation.

On the other hand, it is also possible to insert the extension 69b into four second tubes 52 at the same time using the integrated precharge tube shown in FIG.

Hereinafter, the automatic extraction of the biological material according to the present invention will be described schematically.

FIG. 12 is a flowchart schematically illustrating a process of performing an automatic extraction operation using the automatic extraction device of a biological material according to the present invention described with reference to FIGS. 1 to 11. In the example shown in the drawings, a process of extracting a gene from a biological material will be described, but the present invention is not limited thereto.

As a preliminary preparation for performing the gene extraction process, the precharge tube 61 is filled with a predetermined amount of the cleaning solution and the elution solution in the predetermined isolation spaces 63, 64, 65, and the sealing tape is attached to the main body 68. ) To the top of the The precharge tube 61 is suspended on a tube holder 85 placed on the table 81 shown in FIG. 8B. On the other hand, the rack 21 in the rotor 10 is mounted with a tube holder, and the first to third tubes 51, 52, 53 are mounted in advance in the tube holder.

Referring to FIG. 12, a pretreatment step 100 of a sample to be extracted from a biological material is performed. For example, in the gene extraction process, the pre-treatment step 100 may add a pre-treatment reagent for gene extraction to a sample such as biological tissue, cells, blood, and the like to dissolve various biological substances, and then add the gene ( Nucleic acid) binding reagent buffer.

After the pretreatment process is finished, the pretreated sample is sucked using a pump or pipette (not shown) provided in the robot 82, and then moved to the first tube 51 on the tube holder 31 to transfer the pretreated sample to the first sample. Inject into the tube (51). At this time, the sample is separated into the supernatant and the lower layer in the first tube 51. The supernatant is needed for the subsequent process, so use a pipette or the like to take the supernatant. (Step 120).

The supernatant of the sample taken by the pipette moves to the top of the second tube 52 and is injected into the second tube 52. Next, the precharge tube 62 is inserted into the upper portion of the second tube 52 using a gripper (not shown) of the robot 82. (Step 130).

As described above, the isolated spaces 63, 64, 65 of the precharge tube 61 are filled with the cleaning liquid and the eluent. In order to inject the cleaning liquid filled in the precharge tube 61 into the second tube 52, the bottom of the first isolation space 63 of the precharge tube 61 is drilled. Perforation may be made by a puncture needle installed in the robot 83. After the perforation has been made, the primary cleaning is performed by performing a centrifugal action in a state in which the cleaning liquid filled in the first isolation space 63 is injected into the second tube 52. During centrifugation, the liquid in the second tube 52 is discharged to the outside through the opening 52a, and the gene is left in the filter 55 (step 140).

Meanwhile, the precharge tube 61 may be replaced with the coupleable precharge tube 90 and the integrated precharge tube described with reference to FIGS. 9A to 11.

Next, the centrifugal action is stopped, and the bottom of the second isolation space 64 of the precharge tube 62 is punctured. After the perforation is made, the secondary cleaning is performed by performing a centrifugal action in a state in which the cleaning liquid filled in the second isolation space 64 is injected into the second tube 52. The liquid used for washing is again discharged to the outside through the opening 52a, and the gene is left in the filter 55 (step 150).

After the second wash, the centrifugal action is stopped. Next, with the precharge tube 61 fitted in the upper portion of the second tube 52, the second tube 52 is inserted in the upper portion of the third tube 53 (step 160).

Then again using the puncture needle to puncture the bottom of the third containment space 65 of the precharge tube 61 so that the elution solution that was filled in the third containment space 65 is injected into the second tube 52. do. The elution solution elutes the genes present in the filter 55. Next, when centrifugation is performed, the eluent containing the gene is carried out to the third tube 53 through the opening 52a of the second tube 52. (Step 170)

The above gene extraction process is just one example that can be performed with the apparatus of the present invention, and different processes may be performed depending on the extraction target.

Referring to FIG. 13, another example of an extraction method using a rotor assembly of a centrifugal separator for automatic extraction of biological material is shown.

Referring to the drawings, an extraction preparation step 200 of biological material is carried out, which can be carried out using the rotor assembly 1 according to the invention described above. That is, in the rotor assembly, the rack 21 is installed to be tilted by the centrifugal force generated when the rotor assembly is rotated, and the tube insertion space provided in the rack 21 or the tube holder 31 accommodated in the rack. The first to third tubes 51, 52, 53 are inserted into the second tube 52, the second tube 52 is provided with a filter 55, and an opening 52a is formed at the bottom of the second tube 52. Refill each of the reagents in the first to third isolation spaces 63, 64 and 65 formed in the precharge tube 61 which can be fitted to the upper end, or prefilled prefill tube tube holder 85 Posted in

Next, as a pretreatment step 210 of the biological material, pretreatment reagents are added to the sample of the biological material to cell lysis, remove cell debris, or remove the sample from the cell or tissue membrane to separate DNA. Depending on the type (bacteria, tissue, etc.), the sample is placed in the first tube and centrifuged to separate the upper and lower layers.

Next, a step (210) of injecting some or all of the pretreated sample into the second tube (52) and inserting the precharge tube (61) into the upper end of the second tube (52). Thereafter, a binding step 230 is performed in which the biological assembly is attached to the filter 55 provided in the second tube 52 by centrifugation using the rotor assembly.

Next, the first cleaning step is performed by centrifugation using the rotor assembly while injecting the reagent in the first isolation space into the second tube 52 by drilling the first isolation space 63 of the prefilled tube. Perform 240. Secondly performing centrifugation using the rotor assembly while injecting the reagent in the second isolation space into the second tube 52 by drilling the second isolation space 64 of the precharge tube 62. A cleaning step 250 takes place. In addition, by inserting the second tube 52 in the state where the precharge tube 61 is inserted into the third tube 53 and drilling the third isolation space, the reagent in the third isolation space is transferred to the second tube. Performing centrifugation using the rotor assembly in a state flowing through the opening at the bottom of the third tube 53, step 260.

Referring to FIG. 14, another example of an extraction method using a rotor assembly of a centrifugal separator for automatic extraction of biological material is shown.

Referring to the drawings, similar to the example of FIG. 13, an extraction preparation step 300 of biological material is performed. Exceptionally, four containment spaces are formed in the precharge tube used in the example shown in FIG. 13, and one of the containment spaces is filled with a binding reagent. Alternatively, or in parallel, the extraction preparation step 300 of the biological material may be performed by placing the prefilled prefilled tube in the tube holder 85.

A pretreatment step 310 of the biological sample is then performed, which adds a pretreatment reagent to the sample of biological material to destroy the cells of the membrane of the cell or tissue, remove cell debris, Therefore, the sample is put into the first tube and separated to separate the upper and lower layers, and the step 320 is performed by injecting the pretreated sample into the second tube and inserting the prefilled tube into the upper end of the second tube 52. .

Next, after injecting the binding reagent contained in the first containment space into the second tube 52 by puncturing the first containment space of the precharge tube, dielectric material is introduced into the filter of the second tube while rotating the rotor assembly. Binding step 330 is performed to attach to 55.

Next, the first cleaning step 340 is performed by puncturing the second containment space of the precharge tube 61 to perform centrifugation using the rotor assembly while injecting the reagent of the second containment space into the second tube. Is performed. Next, a second cleaning step 350 is performed in which centrifugation is performed using the rotor assembly while perforating the third containment space of the prefilled tube while injecting reagent in the third containment space into the second tube.

Subsequently, the second tube with the precharge tube inserted is inserted into the third tube 53, and the reagent in the fourth isolation space is transferred to the bottom of the second tube 52 by drilling the fourth isolation space. Step 360 is performed, where centrifugation is performed using the rotor assembly with flow through the opening 52a in the third tube 53.

In Figures 13 and 14 the transfer of the tube, perforation of the prefilled tube, suction of the sample by the pump or pipette and injection into the tube, centrifugation by the rotor assembly, etc. can be made as described with reference to Figure 12. have.

1. Rotor Assembly 10. Rotor
11.Circumferential wall 12. Partition
13. Rotating shaft insertion hole 14. Bulkhead

Claims (11)

A rotor having a partition wall for dividing an inner space into a plurality of divided spaces and a rotation shaft insertion hole formed in a center of the inner space;
A plurality of racks removably received within the divided spaces of the rotor, the plurality of racks being inclined upon rotation of the rotor and forming a plurality of tube insertion spaces;
A plurality of tubes, which can be removably inserted into a plurality of tube insertion spaces provided in the plurality of racks; And
And a prefilled tube that can be removably fitted on top of the plurality of tubes. The method of claim 1,
And the plurality of tube insertion spaces are formed in a tube holder that can be removably received in each of the plurality of racks. 3. The method according to claim 1 or 2,
The swing shaft protrudes from the partition wall, and the swing shaft is coupled to the shaft insertion groove formed in the side wall of the rack, whereby the rack rotates about the swing shaft and tilts when the rotor assembly rotates. Rotor assembly of centrifugal separator for extraction. 3. The method according to claim 1 or 2,
At least one of the plurality of tubes is a rotor assembly of the centrifuge device for the automatic extraction of biological material, the filter is provided inside the tube and the bottom is open. 3. The method according to claim 1 or 2,
The precharge tube includes a body portion having at least one isolation space and an extension portion extending from the body portion and having an opening at a bottom thereof, and perforating a bottom portion of the body portion corresponding to each bottom of the at least one isolation space. The rotor assembly of the centrifugal separator for the automatic extraction of biological material, wherein a solution filled in each isolation space can be discharged through the opening of the extension. 3. The method according to claim 1 or 2,
And a bearing for rotatably supporting the upper end of the rotor and a support member provided with the bearing, wherein the rotor assembly of the centrifugal separator for automatic extraction of biological material. 3. The method according to claim 1 or 2,
The precharge tube may include a main body portion having at least one isolation space, an extension portion extending from the main body portion and having an opening at a bottom thereof, a coupling protrusion and a coupling recess formed on outer surfaces of the main body portions opposite to each other; and,
The joining protrusions or joining recesses of one prefilling tube may be joined or separated from each other with the joining recesses or joining protrusions of the other prefilling tube,
Of the centrifugal device for automatic extraction of biological material, by which a solution filled in each isolation space can be discharged through the opening of the extension by puncturing the bottom of the body portion corresponding to each bottom of the at least one isolation space. Rotor assembly. 3. The method according to claim 1 or 2,
In order to measure the amount of rotation of the rotor assembly, a camera capable of capturing markings marked on the rotor assembly or a light receiving sensor capable of receiving light of a light emitting portion installed in the rotor assembly, the automatic extraction of biological material Rotor assembly of centrifugal separator for the. 3. The method according to claim 1 or 2,
A chamber disposed outside the rotor to accommodate the rotor therein;
And a fan or cooling device capable of blowing cooling air into the chamber, wherein the rotor assembly of the centrifugal separator for automatic extraction of biological material. In a rotor assembly having a rack installed in a plurality of divided spaces formed by the partition wall, the rack is installed so that the rack can be tilted by centrifugal force generated when the rotor assembly rotates, and in the tube insertion space or the rack provided in the rack A first to third tube formed in a prefilled tube that can insert first to third tubes into a received tube holder, provide a filter in the second tube, form an opening in the bottom, and fit into an upper end of the second tube; Extracting a biological material, either filling each of the reagents in the sequestration spaces or placing a prefilled prefilled tube in a tube holder;
In order to separate DNA by adding a pretreatment reagent to a sample of biological material, cells of the membrane of cells or tissues are removed (cell lysis), cell debris is removed, or the sample is placed in a first tube and centrifuged according to the sample type. Pretreatment of the biological sample to separate the upper and lower layers;
Injecting some or all of the pretreated sample into the second tube and inserting the precharge tube into an upper end of the second tube;
Binding to attach a biological dielectric material to a filter provided in a second tube by performing centrifugation using the rotor assembly;
Performing a centrifugal separation using the rotor assembly while injecting reagents in the first containment space into the second tube by puncturing the first containment space of the prefilled tube;
A second cleaning step of performing centrifugation using the rotor assembly while injecting reagent in the second isolation space into the second tube by puncturing the second isolation space of the precharge tube;
Inserting the second tube with the prefilled tube inserted into the third tube and puncturing the third containment space, the reagent in the third containment space passes through the opening at the bottom of the second tube to the third tube. Performing centrifugation using the rotor assembly in a flowing state, comprising: a rotor assembly of the centrifugal separator for automatic extraction of biological material. In a rotor assembly having a rack installed in a plurality of divided spaces formed by the partition wall, the rack is installed so that the rack can be tilted by centrifugal force generated when the rotor assembly rotates, and in the tube insertion space or the rack provided in the rack A first to third tube formed in the prefilled tube that can insert first to third tubes into the received tube holder, provide a filter in the second tube, form an opening in the bottom, and fit into the top of the second tube; Extracting the biological material, either filling each of the reagents in the sequestration spaces or placing a prefilled prefilled tube in the tube holder;
Cell lysis of cells or tissue membranes by adding pretreatment reagents to a sample of biological material, removal of cell debris, or separation of the upper and lower layers by placing the sample in a first tube and centrifuging according to the sample type For pretreatment of the biological sample;
Injecting some or all of the pretreated sample into the second tube and inserting the precharge tube into an upper end of the second tube;
A binding step of injecting a binding reagent contained in the first isolation space into the second tube by puncturing the first isolation space of the precharge tube, and then attaching the dielectric material to the filter of the second tube while rotating the rotor assembly;
Performing a centrifugal separation using the rotor assembly while injecting reagents in the second containment space into the second tube by puncturing the second containment space of the prefilled tube;
Performing a centrifugal separation using the rotor assembly while injecting the reagent of the third isolation space into the second tube by puncturing the third isolation space of the precharge tube;
Inserting the second tube with the prefilled tube inserted into the third tube and puncturing the fourth containment space, the reagent in the fourth containment space passes through the opening at the bottom of the second tube to the third tube. Performing centrifugation using the rotor assembly in a flowing state; extracting the rotor assembly of the centrifugal apparatus for automatic extraction of biological material.

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