Centrifugal reaction device and centrifugal reaction method

Abstract

The centrifugal reactor comprises a centrifugal disk having a centrifuge shaft and at least one centrifuge holder, the centrifuge holder being annularly arranged around the centrifuge shaft, and at least one magnetic block arranged on at least one side of the centrifuge holder. The centrifuge holder detachably holds reaction tubes, which contain magnetic beads. The magnetic beads move within the reaction tubes under the combined force of the magnetic force of the magnetic block and the centrifugal force. Therefore, the reaction mixture is mixed quickly and thoroughly to promote the reaction. The magnetic beads adsorb the product, causing the valves of the reaction tubes to open under the centrifugal force, allowing waste liquid to be discharged, reducing the consumption of consumables in the reaction tubes.

Description

[Technical field]

[0001] The present disclosure relates to a reaction device and a reaction method for experimental purposes, and more particularly to a centrifugal reaction device and a centrifugal reaction method that are effective for performing molecular biological assays with one touch under centrifugal force and magnetic force. [Background technology]

[0002] Advances in biotechnology have led to the development of biochemical and molecular biological There is an increasing demand to perform assays with magnetic biological methods. Existing commercially available devices for performing nucleic acid extraction using magnetic beads fall into two categories: 1. Magnetic bar The magnetic bar and its outer magnetic bar sleeve move up and down while rotating in the reaction vessel, stirring the reaction mixture. The reaction mixture, magnetic beads and magnetic bar sleeve are then taken out of the reaction vessel together to be transferred to another reaction vessel for the next reaction. The magnetic beads and magnetic bar sleeve must be continuously moved in and out of the reaction vessel, which lengthens the treatment process and leads to contamination risks. In addition, the magnetic bar sleeve and reaction vessel used in the reaction process are consumables and need to be replaced as necessary, which increases costs. 2. Magnetic Block With the magnetic blocks around and the magnetic beads inside the micropipette, the micropipette moves the solution back and forth vertically to mix the solution and cause a reaction. In order to move the reaction mixture to a different reaction tank and react, the magnetic beads need to be attracted to the micropipette by the surrounding magnetic blocks. The magnetic beads and the micropipette need to be continuously moved in and out of the reaction tank, which lengthens the processing process and makes large-scale processing impossible. In addition, the special micropipette and reaction tank used in the reaction process are consumables and need to be replaced as needed, which increases costs.

[0003] Therefore, there is a need to provide an extraction device or analyzer that operates quickly and efficiently and reduces costs when there are a large number of samples or specimens. However, existing commercially available extraction devices or analyzers are slow to operate, contain a large number of consumables, and cannot perform all molecular biological reactions in one touch. Summary of the Invention

[0004] Considering the above-mentioned shortcomings of the prior art, the objective of the present disclosure is to provide a centrifugal reaction apparatus. Due to the design of the magnetic block, during the centrifugation process, the magnetic beads in the reaction tube are subjected to not only the centrifugal force but also the magnetic force of the magnetic block. The magnetic beads move in the reaction tube under the combined force of the magnetic force of the magnetic block and the centrifugal force. The movement of the magnetic beads causes the reaction mixture in the reaction tube to move in various directions and be thoroughly mixed. Furthermore, the reaction tube is designed such that the check valve can be opened by the centrifugal force, allowing the magnetic beads to adsorb the product and discharge the waste liquid. Therefore, the centrifugal reaction apparatus is advantageous in that the extraction, reaction, washing and / or signal assay are performed in the same reaction microtube, reducing the quantitative requirement for the reaction tube.

[0005] In order to achieve another object of the present disclosure, an embodiment of the present disclosure comprises: A centrifugal disk comprising a centrifugal shaft and at least one centrifugal holder, the centrifugal holder comprising: a centrifugal disk arranged annularly around the centrifugal shaft; at least one magnetic block disposed on at least one side of the centrifuge holder; the centrifuge holder removably holds a reaction tube, the reaction tube containing magnetic beads; A centrifugal reaction device is provided in which the magnetic beads move within the reaction tube by receiving the sum of the magnetic force of the magnetic block and the centrifugal force.

[0006] Preferably, the magnetic block and the centrifugal disk are not connected and therefore do not move together.

[0007] Preferably, the magnetic block is an electromagnet, a magnet, or a combination thereof.

[0008] Preferably, the magnetic block and the centrifugal disk are connected and therefore move together.

[0009] Preferably, the electromagnet is disposed within the magnetic block.

[0010] Preferably, the magnetic block and the centrifugal holder are spaced apart from each other by a distance of 0 to 300 mm.

[0011] Preferably, the centrifugal force is from 1 to 80,000 g.

[0012] Preferably, the magnetic force of the magnetic block is 1 to 15,000 Gauss.

[0013] Preferably, the reaction tube comprises at least one reaction zone and at least one check valve.

[0014] Preferably, the check valves of the reaction tubes are electrically or magnetically controlled to open and close.

[0015] Preferably, the magnetic beads are capable of binding biological molecules, including antibodies, aptamers, peptides and nucleic acids. The nanotubes are coated with cellulose acetate molecules.

[0016] For further purposes of the present disclosure, one embodiment thereof comprises: introducing a sample and / or a reaction reagent into at least one reaction zone of a reaction tube in a centrifugal reaction device to form a reaction mixture; Introducing magnetic beads into a reaction region; A step of performing a centrifugation process in the reaction tube by receiving a first centrifugal force to start a first reaction, in which the magnetic beads move in the reaction tube by receiving a sum of the magnetic force of the magnetic block and the centrifugal force; and performing a centrifugation process in the reaction tube under a second centrifugal force greater than the first centrifugal force to open the first check valve, thereby discharging a waste liquid of the first reaction.

[0017] Preferably, the method further comprises the step of introducing a wash buffer to remove impurities.

[0018] Preferably, the method further comprises the step of recovering the product from the magnetic beads.

[0019] Preferably, the magnetic beads are coated with biomolecules including antibodies, aptamers, peptides or nucleic acids.

[0020] Preferably, the magnetic beads are coated with silicon dioxide.

[0021] Preferably, the product is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0022] Preferably, a polymerase chain reaction can be carried out in the reaction area.

[0023] Preferably, nucleic acid hybridization can be carried out in the reaction area.

[0024] The above and other objects, features, and advantages of the present disclosure will become apparent with reference to the following description, preferred embodiments, and accompanying drawings. [Brief description of the drawings]

[0025] The following detailed description includes sufficient specific details to allow a complete understanding of the embodiments of the present disclosure. However, it is apparent that one or more embodiments may be practiced without the specific details. In other circumstances, for the sake of brevity, well-known structures and process flows are generally shown in the accompanying drawings.

[0026] [Figure 1] FIG. 1 is a top view of a centrifugal reactor according to a first embodiment of the present disclosure.

[0027] [Diagram 2]FIG. 1 is a cross-sectional view of a centrifugal reactor according to a first embodiment of the present disclosure.

[0028] [Diagram 3] FIG. 2 is a cross-sectional view of a centrifugal reactor according to a second embodiment of the present disclosure.

[0029] [Figure 4] FIG. 13 is a top view of a centrifugal reactor according to a third embodiment of the present disclosure.

[0030] [Diagram 5] FIG. 11 is a cross-sectional view of a centrifugal reactor according to a third embodiment of the present disclosure.

[0031] [Figure 6] FIG. 11 is a cross-sectional view of a centrifugal reactor according to a fourth embodiment of the present disclosure.

[0032] [Figure 7] FIG. 1 is a schematic diagram showing how magnets are arranged in the outer magnetic block according to the first embodiment of the present disclosure. A and A' are connected, and the three-dimensional structure of the outer magnetic block is cylindrical. The magnets are arranged in the solid core block, but not in the hollow core block. The arrows indicate the movement direction of the magnetic beads.

[0033] [Figure 8] FIG. 1 is a schematic diagram showing how magnets are arranged in the inner and outer magnetic blocks according to the first embodiment of the present disclosure. The outer magnetic block surrounds the outside, and the inner magnetic block surrounds the inside. Magnets are arranged in the solid core block, but not in the hollow core block. The arrows indicate the direction of magnetic bead movement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The embodiments of the present disclosure are described herein with reference to the accompanying drawings. The embodiments may be implemented in different forms, and this should not be construed as imposing any limitations on the implementation or application of the particular embodiment of the subject matter claimed by the present disclosure. The present embodiments disclose features of the particular embodiments, as well as steps and sequences of methods for constructing and operating the particular embodiments. However, the same or equivalent functions and sequences of steps may be achieved by any other particular embodiment. The present embodiments enable the disclosure to be fully and completely disclosed so as to fully convey the gist of the disclosure to those skilled in the art. Similar components shown in the accompanying drawings are designated with similar reference numerals / characters. Well-known functions and structures are not described in detail herein for the sake of brevity.

[0035] Unless otherwise defined, all technical terms and terminology used herein are generally understandable to those of ordinary skill in the art. In the event of any discrepancy between the meaning of a technical term or terminology and the understanding of such a term by those of ordinary skill in the art, the present specification (including definitions) shall control.

[0036] Each singular noun as used herein shall be construed to include the plural of the noun, and each plural noun as used herein shall be construed to include the singular of the noun, unless the result is contradictory to the context. Furthermore, as used herein, the expressions "at least a," "at least one," and "one or more" have the same meaning and mean "one, two, three or more."

[0037] As used herein, the conjunction "consisting essentially of" is used to define a composition, method, or apparatus that includes materials, steps, features, ingredients, or components other than those expressly defined herein, and is limited to use scenarios where the additional materials, steps, features, ingredients, or components do not significantly affect the essential and novel characteristics of the claimed invention. The scope of the subject matter of the conjunction "consisting essentially of" lies between the scope of the subject matter of the conjunction "comprises" and the scope of the subject matter of the conjunction "consisting of."

[0038] All numerical ranges and parameters used herein are intended to be approximate, however, the relevant numerical values ​​disclosed in certain embodiments are presented herein as precisely as possible. However, numerical values ​​inherently entail standard deviations resulting from individual testing methods. The adjective "approximate" as used herein generally refers to the fact that a numerical value disclosed in a particular embodiment is in reality up to 10%, up to 5%, up to 1%, or up to 0.5% greater or less than the numerical value disclosed in a particular embodiment. Alternatively, the adjective "approximate" as used herein generally refers to the fact that a numerical value actually falls within an acceptable standard deviation of the mean, depending on design choices made by those skilled in the art. Unless otherwise indicated, all ranges, numbers, values, and percentages disclosed herein (e.g., descriptions of amounts of materials used, durations, temperatures, operating standards, ratios, etc.) are intended to be approximate and are therefore not limiting to the embodiments. Thus, unless stated to the contrary, all numerical values ​​and parameters disclosed herein are approximate and are subject to change as necessary. At the very least, the numerical values ​​and parameters are to be construed in accordance with conventional rules of significant figures and numerical systems. Numeric ranges disclosed herein are defined from each endpoint to the other endpoint, or between two endpoints. Unless otherwise specified, numerical ranges disclosed herein include each endpoint.

[0039] One embodiment provides a centrifugal reaction device comprising a centrifuge disk and at least one magnetic block. The centrifuge disk comprises a centrifuge shaft and at least one centrifuge holder. In a preferred embodiment, the centrifuge shaft is disposed at the center of the centrifuge disk. When a driving force is transmitted to the centrifuge shaft to rotate the centrifuge disk, the centrifuge disk remains stable and does not shift. In one embodiment, the centrifuge holder is disposed annularly around the centrifuge shaft and moves in response to the rotation of the centrifuge disk.

[0040] In one embodiment, the magnetic block is disposed on at least one side of the centrifuge holder. In one embodiment, the magnetic block is disposed on the inside, outside, upper, lower, side, or combination thereof of the centrifuge holder. In a specific embodiment, the magnetic blocks disposed on the inside, outside, and side of the centrifuge holder are on a horizontal plane passing through the center of the reaction tube, while the magnetic blocks disposed on the upper and lower sides of the centrifuge holder are above and below the horizontal plane of the reaction tube, respectively.

[0041] In one embodiment, the centrifugal holder of the centrifugal reaction device detachably holds the reaction tube, and the reaction tube contains magnetic beads. In one embodiment, the magnetic beads move in the reaction tube under the force of the sum of the magnetic force of the magnetic block and the centrifugal force. Therefore, the direction and speed of the movement of the magnetic beads depend on the force that the magnetic beads are subjected to, i.e., the sum of the magnetic force and the centrifugal force. As used herein, the expression "direction of the magnetic force of the magnetic block toward the magnetic beads" refers to the direction of the force that the magnetic field of the magnetic block exerts on the magnetic beads. As used herein, the expression "magnitude of the magnetic force of the magnetic block toward the magnetic beads" refers to the magnitude of the force that the magnetic field of the magnetic block exerts on the magnetic beads. In a preferred embodiment, a change in the direction or magnitude of the magnetic force of the magnetic block toward the magnetic beads occurs.

[0042] In one embodiment, the reaction tube is placed in a centrifuge holder and moves with the centrifuge holder. In a preferred embodiment, the centrifuge holder removably holds the reaction tube. Samples or reagents are manually or mechanically introduced into the reaction tube, and then the reaction tube is attached to the centrifuge holder for the centrifugation process. The centrifugation process ends only when the reaction is complete. The end of the centrifugation process is not followed by removal of the reaction tube, but by assay or recovery of the product. Alternatively, the end of the centrifugation process is followed by manual or mechanical removal of the reaction tube from the centrifuge holder, followed by assay or recovery of the product. In a feasible embodiment, the reaction tubes each have a supply port and a distribution port connected to a supply tube and a distribution tube, respectively. Samples or reagents are introduced into the reaction tube through the supply port after passing through the supply tube. Waste liquid or products are discharged from the reaction tube through the distribution port after passing through the distribution port. In this situation, the reaction tube is not necessarily removed from the centrifuge holder. In one embodiment, the reaction tube contains magnetic beads, which move in the reaction tube under the influence of centrifugal and magnetic forces.

[0043] The present disclosure does not limit the material, shape or volume of the reaction tube, as long as the movement of the magnetic beads, the reaction and operation of the centrifuge device are not affected. In one embodiment, the material from which the reaction tube is made is plastic, glass or steel. The plastic from which the reaction tube is made includes polyethylene (PE), polycarbonate (PC), polypropylene (PP), etc., and is preferably polypropylene. In a preferred embodiment, the reaction tube is made of a light-transmitting and highly transparent material, more preferably a nearly transparent material, to facilitate assaying or monitoring the reaction intermediate or final product in the reaction tube. In one embodiment, the reaction tube has a volume of 500 mL, 250 mL, 50 mL, 15 mL, 2 mL, 1.5 mL, 0.65 mL, or 0.2 mL. In one embodiment, the reaction tube is, for example, a conical centrifuge tube, a flat-bottom centrifuge tube or a round-bottom centrifuge tube, as long as the shape of its bottom is designed differently to meet the experimental needs.

[0044] In one embodiment, the magnetic blocks and the centrifuge disk are not necessarily connected, and therefore do not necessarily move together. Therefore, the magnetic blocks and the centrifuge disk are not connected, and the magnetic blocks do not move even when the centrifuge disk rotates. The magnetic blocks are arranged inside, outside, on the upper side, on the lower side, or a combination thereof, of the centrifuge holder. In this embodiment, the magnetic blocks are not arranged on the sides of the centrifuge holder so as not to interfere with the rotation of the centrifuge holder. In one embodiment, the magnetic blocks are electromagnets, magnets, or a combination thereof. The present disclosure is not limited to the type and shape of the magnets or electromagnets, as long as the magnets or electromagnets generate a magnetic force large enough to attract the magnetic beads. In one embodiment, after the reaction tube rotates and faces different directions, the number of magnets is not evenly arranged in different directions. For example, if the horizontal plane passing through the center of the reaction tube is taken as the reference plane, when the magnet is on the horizontal plane, an angle exists between the direction of the magnetic force of the magnet and the horizontal plane, and the magnetic beads will shift upward away from the horizontal plane. Conversely, if the magnet is below the horizontal plane, there will be an included angle between the direction of the magnet's magnetic force and the horizontal plane, which will shift the magnetic beads downward from the horizontal plane. The magnet will be oriented in different directions relative to the centrifuge disk, so that the horizontal or vertical position will change, allowing the magnetic beads to move vertically and horizontally within the reaction tube. Thus, the specific position of the magnetic block can help to adjust the magnetic force of the magnet toward the magnetic beads, thereby controlling how the magnetic beads move. In one embodiment, the magnetic force is controlled by activating or deactivating an electromagnet. Thus, the presence, strength, and direction of the magnetic force depend on whether or not a current passes through the electromagnet, and the magnitude of that current. The electromagnet controls the movement of the magnetic beads in the same way as a magnet.

[0045] In one embodiment, each magnetic block and the centrifuge disk are connected to each other and move together. Thus, each magnetic block and the centrifuge disk are connected to each other and move with the rotation of the centrifuge disk. The magnetic blocks are arranged inside, outside, above, below, or on the side of the centrifuge holder, or a combination thereof. The magnetic blocks are arranged on the side of the centrifuge holder without interfering with the rotation of the centrifuge holder. In one embodiment, the magnetic blocks are electromagnets. The working principle and function of the electromagnets have been described above.

[0046] In one embodiment, each magnetic block and the centrifugal holder are separated by a distance of 0-300 mm, preferably 0-20 mm, more preferably 0-3 mm. If the distance between each magnetic block and the centrifugal holder is too large, the magnetic force is insufficient to attract the magnetic beads, and the movable range of the magnetic beads is limited. In order to prevent wear and damage caused by contact friction between components due to the mismatch of the movement mode between each magnetic block and the centrifugal disk, each magnetic block is not connected to the centrifugal disk and does not move together with the centrifugal disk, and therefore each magnetic block and the centrifugal holder are separated by a distance of preferably less than 10 mm, more preferably less than 5 mm.

[0047] The centrifugal disk of the centrifugal reactor can be an existing commercially available centrifugal device. The present disclosure does not limit the magnitude of the centrifugal force, as long as an appropriate centrifugal device is selected as necessary and its centrifugal force is adjusted. In one embodiment, the centrifugal force is 0 to 80,000 g, preferably 0 to 12,000 g, more preferably 0 to 6,000 g. In a feasible situation, the reaction tube is an existing commercially available centrifuge tube and can sufficiently mix the reaction mixture. In a preferred embodiment, the reaction tube of the present disclosure is controlled to open and close the check valve according to the magnitude of the centrifugal force to automatically discharge the waste liquid without human intervention.

[0048] The magnetic beads of the centrifugal reaction device are provided in the form of existing commercial products. In a feasible embodiment, the magnetic block is additionally arranged on the existing centrifugal device. The magnetic block is fixed to the centrifugal device but does not move together with the centrifugal disk. Alternatively, the magnetic block is fixed to the centrifugal device and moves together with the centrifugal disk. In one embodiment, the magnetic block generates a magnetic force of 1 to 15,000 Gauss, preferably 1 to 12,000 Gauss, more preferably 1 to 8,000 Gauss. In a feasible embodiment, the magnetic forces of the magnetic blocks are of the same or different magnitudes.

[0049] In one embodiment, each reaction tube includes at least one reaction area for receiving a reaction mixture including reaction reagents, washing liquids, and / or samples, and at least one check valve arranged in the centrifugal direction of the reaction area. When the centrifugal speed or force exceeds a threshold, the check valve opens, allowing the reaction mixture to flow away from the axis of rotation under the centrifugal force and exit the reaction area. When the centrifugal speed or force is less than a threshold, the check valve closes, preventing the reaction mixture from flowing back to the original reaction area. The check valve selectively opens and closes repeatedly, allowing waste liquid to be discharged multiple times during the course of the reaction. Furthermore, the reaction tube may be divided into multiple reaction areas, in which reactants consisting of biological molecules such as proteins and nucleic acids can undergo reactions such as purification, amplification, and analysis.

[0050] The "check valve" is a backflow prevention mechanism that controllably retains reaction reagents, washing fluid, and / or specimens in a reaction area before the start of the centrifugation process, and simultaneously with the start of the centrifugation process, moves the reaction reagents, washing fluid, and / or specimens in one direction through the "check valve" to reach another reaction area.

[0051] Check valves operate on different principles. For example, check valves can be mechanical check valves with springs of different elastic constants and balls of different weights. Mechanical check valves can be metallic or non-metallic. Different magnitudes of centrifugal force correspond to valves with different elastic constants, such that different valves in a reaction microtube open and close under different magnitudes of centrifugal force. Due to the different magnitudes of centrifugal force, the check valve can control the retention or discharge of the reaction mixture. Furthermore, the check valve can be electrically or magnetically controlled to open and close the check valve.

[0052] For example, if the opening and closing of a check valve is controlled by a compression spring, at rest, in the absence of any centrifugal process or centrifugal force creating a compressive stress on the spring, the compression spring will fully extend, resulting in a fully closed check valve. When the centrifugal force reaches the compressive stress of the spring, the gradually increasing magnitude of the centrifugal force will compress and gradually shorten the compression spring, thus opening the check valve, thereby allowing the reaction mixture to move away from the axis of rotation and through the check valve.

[0053] All waste liquids generated during the reaction process are collected in an airtight or vacuum state depending on the amount of waste liquid or whether the reaction microtube has an opening. In one embodiment, the waste liquid is collected in an airtight state under the condition that the centrifugal reaction microtube further comprises a waste liquid area, and the check valve and the reaction area are separated so that the reaction mixture or washing liquid generated during the reaction leaves the reaction area as a result of the centrifugation process and reaches the waste liquid area without returning.

[0054] In one embodiment, the magnetic beads are coated with biomolecules and adapted to allow the reaction mixture to undergo hybridization or adsorption. Biomolecules include antibodies, aptamers, peptides or nucleic acids.

[0055] The outside of each reaction zone is adjacent to a heating module for increasing the temperature of the reaction zone by substantial contact heating. Preferably, the heating module is U-shaped or O-shaped to effectively surround the reaction zone, thereby accelerating the heating of the reaction zone 111. When the reaction tube is divided into multiple reaction zones, it is feasible to arrange a heating module on at least one side of each reaction zone to independently control the reaction temperature of each reaction zone. In a feasible embodiment, the centrifugal device includes an inner magnetic base, but its outer magnetic base is replaced by a heating module.

[0056] In one embodiment, the centrifugal reactor further comprises a temperature control module for controlling the reaction temperature in the centrifugal reaction microtube via a heating block, liquid (e.g., hot water), gas (e.g., hot air), or far-infrared radiation. Preferably, the heating block and liquid are arranged in a U-shape or O-shape to surround the centrifugal reaction microtube during the heating process to enhance the efficiency of temperature control.

[0057] In one embodiment, the centrifugal reaction device may further comprise a signal assay module for assaying the reaction signal of the centrifugal reaction microtube. For example, the signal assay module is a fluorescence, photoluminescence or visible light photo sensing system. When the reaction in each reaction area of ​​the centrifugal reaction microtube is completed, a biomolecule (such as an antibody, an aptamer, a peptide or a nucleic acid) for use in marker fluorescence, photoluminescence or color signal is added to each reaction area to assay the signal, thereby performing a qualitative or quantitative analysis of the reaction product.

[0058] In one embodiment, there are various options for applying force to the magnetic beads, including applying only magnetic force, only centrifugal force, or both magnetic and centrifugal forces to the magnetic beads. For illustrative purposes, a particular situation of relevance is described below. ● The centrifuge disk does not rotate, and the magnetic block generates a magnetic force specifically for the magnetic beads. ● As the centrifuge disk rotates, the magnetic block generates a magnetic force whose magnitude is close to zero or negligible, with the magnetic beads only subjected to the centrifugal force. ● The centrifuge disk rotates, the magnetic block generates a magnetic force, and the magnetic beads are subjected to both magnetic and centrifugal forces. In one embodiment, the option for applying force to the magnetic beads depends on the timing. In one embodiment, the centrifuge disk moves up, moves down and rotates relative to the magnetic block to change its position relative to the magnetic block. In one embodiment, the centrifuge disk does not rotate, possibly moves up or down, and the magnetic block rotates and moves up or down to drive the magnetic beads to move and mix the reaction mixture, and then the centrifuge disk rotates to generate centrifugal force that opens the check valve, thereby discharging the waste liquid.

[0059] In another embodiment, the present disclosure provides a centrifugal reaction method comprising the step of introducing a specimen and a reaction reagent into at least one reaction area of ​​a reaction tube in a centrifugal reaction device to form a reaction mixture. In a preferred embodiment, the specimen comprises fresh or frozen whole blood, serum, plasma, bone marrow, umbilical cord blood, cells, urine, cadavers, tissues and cell cultures. In one embodiment, the reaction reagent is a lysis buffer, a binding buffer, a washing buffer or an elution buffer.

[0060] The centrifugal reaction method includes the steps of: introducing magnetic beads into a reaction region; and performing a centrifugation process in the reaction tube under a first centrifugal force so that a first reaction occurs, the magnetic beads moving in the reaction tube under the sum of the magnetic force of the magnetic block and the centrifugal force. In a preferred embodiment, the first reaction includes exposing the nucleic acid in the cell or tissue with a lysis buffer. In a preferred embodiment, the reaction method further includes the step of introducing a binding buffer to promote the binding of the nucleic acid to the magnetic beads. In a preferred embodiment, the reaction method further includes the step of introducing a washing buffer to rinse the nucleic acid, the magnetic beads and the binding substance, as well as to remove salts and impurities. Due to the variation in the direction of the magnetic force acting on the centrifugal holder, the magnetic beads move in the reaction tube to promote sufficient mixing and speed up the mixing process.

[0061] The centrifugal reaction method further includes carrying out a centrifugation process in the reaction tube under the second centrifugal force, opening the check valve, and thereby discharging the waste liquid of the first reaction. When the groove is disposed in the reaction tube, the magnetic beads fall into the groove, and are not discharged from the reaction tube through the open check valve together with the waste liquid. In a possible embodiment, the second centrifugal force is greater than the first centrifugal force.

[0062] The centrifugal reaction method further comprises a step of recovering the product from the magnetic beads. In a preferred embodiment, the reaction method further comprises a step of introducing a washing buffer to rinse and extract the product from the magnetic beads. In one embodiment, the product is recycled from the magnetic beads. In one embodiment, the magnetic beads separated from the product are not removed from the reaction tube, but are attracted to a magnetic block and thus collected in a specific region of the reaction tube. In a preferred embodiment, the concentration of the product is assayed using a spectrophotometer in the centrifugal reaction device.

[0063] To meet experimental needs, the magnetic beads for use in the centrifugal reaction method are coated with biomolecules, including antibodies, aptamers, peptides, or nucleic acids. In a preferred embodiment, the magnetic beads are coated with silicon dioxide. In a feasible embodiment, the product generated in the centrifugal reaction method is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In a feasible embodiment, polymerase chain reaction or nucleic acid hybridization is carried out in the reaction area.

[0064] Specific embodiments of the centrifugal reactor

[0065] As shown in FIG. 1, in the first embodiment, the centrifugal reaction apparatus 1 includes a centrifugal disk 11 and a magnetic block 12. The centrifugal disk 11 includes a centrifugal shaft 111 and a centrifugal holder 112. The centrifugal shaft 111 is disposed at the center of the centrifugal disk 11. To start the centrifugal process, the centrifugal shaft 111 serves as a rotation axis for rotating the centrifugal disk 11. The centrifugal holder 112 is disposed on the centrifugal disk and is disposed so as to surround the centrifugal shaft 111 annularly and symmetrically around the centrifugal shaft 111. The magnetic block 12 is disposed on at least one side of the centrifugal holder 112. The magnetic block 12 disposed inside the centrifugal holder 112 and located close to the centrifugal shaft is an inner magnetic block 121. The magnetic block 12 disposed outside the centrifugal holder 112 and located on the tip side of the centrifugal shaft is an outer magnetic block 122. In the first embodiment, the magnetic block 12 does not move together with the centrifugal disk 11. Therefore, during the centrifugation process, the magnetic block 12 does not move in response to the rotation of the centrifuge disk 11. The centrifugal reaction device 1 may further include a reaction tube 13 and at least one magnetic bead 14. Preferably, the reaction tube 13 is detachably disposed on the centrifuge holder 112. During the centrifugation process, the reaction tube 13 disposed on the centrifuge holder 112 moves in response to the rotation of the centrifuge disk 11. At least one magnetic bead 14 is disposed in the reaction tube. During the centrifugation process, the reaction tube 13 rotates together with the centrifuge disk 11, thereby moving the magnetic bead disposed in the reaction tube 13.

[0066] 2 is a cross-sectional view taken along the dashed line in FIG. 1. As shown in FIG. 2, in the first embodiment, the magnetic block 12 further includes an upper magnetic block 123 and a lower magnetic block 124. The magnetic block 12 arranged above the horizontal surface of the centrifugal holder 112 is the upper magnetic block 123. The magnetic block 12 arranged below the horizontal surface of the centrifugal holder 112 is the lower magnetic block 124. In the first embodiment, the magnetic block 12 does not move together with the centrifugal disk 11. Therefore, the upper magnetic block 123 and the lower magnetic block 124 do not rotate together with the centrifugal disk 11 during the centrifugal process.

[0067] As shown in Fig. 3, in the second embodiment, the magnetic blocks 12 of the centrifugal reaction device 1 do not necessarily have to be arranged on the upper side, lower side, inside, and outside of the centrifugal disk 11. In order to precisely and delicately control the way in which the magnetic beads 14 are moved in the reaction tube 13, the magnetic blocks 12 are arranged on the centrifugal holder 112 and positioned in different directions of the centrifugal holder 112 as necessary around the centrifugal holder 112. In a feasible embodiment, the six magnetic blocks 12 arranged on the centrifugal holder 112 are spaced apart from each other by 60 degrees around the center of the centrifugal holder 112 when viewed from a cross section along a line connecting the centrifugal holder 112 and the rotation axis.

[0068] As shown in FIG. 4, in the third embodiment, the centrifugal reaction device 1 includes a centrifugal disk 11 and a magnetic block 12. The centrifugal disk 11 includes a centrifugal shaft 111 and a centrifugal holder 112. The centrifugal shaft 111 is disposed at the center of the centrifugal disk 11. To start the centrifugal process, the centrifugal shaft 111 functions as a rotation axis for rotating the centrifugal disk 11. The centrifugal holder 112 is disposed on the centrifugal disk 11 and surrounds the centrifugal shaft 111 in an annular shape with the centrifugal shaft 111 at the center. The magnetic block 12 is disposed on at least one side of the centrifugal holder 112. The magnetic block 12 disposed inside the centrifugal holder 112 and located close to the centrifugal shaft is the inner magnetic block 121. The magnetic block 12 disposed outside the centrifugal holder 112 and located on the tip side of the centrifugal shaft 111 is the outer magnetic block 122. The magnetic block 12 arranged on the side of the centrifugal holder 112 is the side magnetic block 125. In the fourth embodiment, the magnetic block 12 moves together with the centrifugal disk 11. Therefore, during the centrifugal process, the magnetic block 12 moves according to the rotation of the centrifugal disk 11. The centrifugal reaction device 1 further includes a reaction tube 13 and at least one magnetic bead 14. The reaction tube 13 is detachably provided on the centrifugal holder 112, and during the centrifugal process, the reaction tube 13 arranged on the centrifugal holder 112 moves according to the rotation of the centrifugal disk 11. At least one magnetic bead 14 is arranged in the reaction tube 13. During the centrifugal process, the reaction tube 13 rotates together with the centrifugal disk 11, so that the magnetic bead arranged in the reaction tube 13 moves.

[0069] FIG. 5 is a cross-sectional view of the centrifugal reaction device taken along the dashed line in FIG. 4. As shown in FIG. 5, in the third embodiment, the magnetic block 12 further includes an upper magnetic block 123 and a lower magnetic block 124. The magnetic block 12 arranged above the horizontal surface of the centrifugal holder 112 is the upper magnetic block 123. The magnetic block 12 arranged below the horizontal surface of the centrifugal holder 112 is the lower magnetic block 124. In this embodiment, the magnetic block 12 moves together with the centrifugal disk 11. Therefore, during the centrifugal process, the upper magnetic block 123 and the lower magnetic block 124 move according to the rotation of the centrifugal disk 11.

[0070] As shown in Fig. 6, in the fourth embodiment, the magnetic blocks 12 of the centrifugal reaction device 1 do not necessarily have to be arranged on the upper side, lower side, inner side, outer side, and side of the centrifugal holder 112. In order to precisely and delicately control the way in which the magnetic beads 14 are moved in the reaction tube 13, the magnetic blocks 12 are arranged on the centrifugal holder 112 and positioned in different directions of the centrifugal holder 112 as necessary around the centrifugal holder 112. In a feasible embodiment, the six magnetic blocks 12 arranged on the centrifugal holder 112 are spaced apart from each other by 60 degrees around the center of the centrifugal holder 112 when viewed from a plane perpendicular to the line connecting the centrifugal holder 112 and the rotating shaft.

[0071] In this embodiment, the magnetic block 12 is a magnet. In a possible embodiment, the magnets arranged in the inner magnetic block 121 or the outer magnetic block 122 are arranged in eight horizontal rows and three vertical columns, numbered in a counterclockwise order as "row m column n" (mn). In a specific embodiment, magnets are arranged in row 1 column 1 (1-1), row 3 column 3 (3-3), row 5 column 1 (5-1), and row 7 column 3 (7-3) of the outer magnetic block, and row 2 column 2 (2-2), row 4 column 2 (4-2), row 6 column 2 (6-2), and row 8 column 2 (8-2) of the inner magnetic block, and no magnets are arranged in the other magnetic blocks. The magnetic beads 14 arranged in the reaction tube 13 move in response to the counterclockwise rotation of the reaction tube 13 and the centrifuge holder. When the reaction tube 13 rotates to face in different directions, the magnetic beads 14 are attracted to the magnets in the inner and outer magnetic blocks facing in different directions. Specifically, when the centrifugal holder 112 rotates counterclockwise, the magnetic beads 14 pass through the magnet in row 1, column 1 (1-1) of the outer magnetic block. For example, the angle between the direction of the magnetic force generated by the magnet (1-1) and the horizontal plane is +30 degrees, which allows the magnetic beads to move outward and upward. Then, the magnetic beads 14 pass through the magnet in row 2, column 2 (2-2) of the inner magnetic block. Since the direction of the magnetic force generated by the magnet (2-2) is parallel to the horizontal plane, the angle between the direction of the magnetic force generated by the magnet (2-2) and the horizontal plane is 0 degrees, which allows the magnetic beads to move inward and downward and approach the horizontal plane. Next, the magnetic beads 14 pass through the magnet in row 3, column 3 (3-3) of the outer magnetic block. The angle between the direction of the magnetic force generated by the magnet (3-3) and the horizontal plane is -30 degrees, which allows the magnetic beads to move outward and downward. Similarly, the magnetic beads move upward, downward, inward, and outward together with the passing magnets. After the reaction tube 13 rotates once and returns to its original position, the magnetic beads 14 perform four reciprocating motions between the inside and outside and two reciprocating motions between the upper and lower sides. The specific arrangement of the magnets arranged in the magnetic block in the above-mentioned situation is shown in Figures 7 and 8.

[0072] In another possible embodiment, the magnets arranged in the outer magnetic block or the inner magnetic block are arranged in six horizontal rows and three vertical columns, numbered in a counterclockwise order as "row m column n" (mn). In a specific embodiment, magnets are arranged in row 1 column 3 (1-3), row 3 column 2 (3-2), and row 5 column 1 (5-1) of the outer magnetic block, and row 2 column 3 (2-3), row 4 column 2 (4-2), and row 6 column 1 (6-1) of the inner magnetic block, but magnets are not arranged in other magnetic blocks. The magnetic beads 14 arranged in the reaction tube 13 move in response to the counterclockwise rotation of the reaction tube 13 and the centrifuge holder. When the reaction tube 13 rotates to face in different directions, the magnetic beads 14 are attracted to the magnets in the inner and outer magnetic blocks facing in different directions. Specifically, when the centrifuge holder 112 rotates counterclockwise, the magnetic beads 14 pass through the magnet in row 1 column 3 (1-3) of the outer magnetic block. For example, the angle between the direction of the magnetic force generated by the magnet (1-3) and the horizontal plane is -30 degrees, and the magnetic beads can move outward and downward. Then, the magnetic beads 14 pass through the magnet in row 2, column 3 (2-3) of the inner magnetic block. The angle between the direction of the magnetic force generated by the magnet (2-3) and the horizontal plane is -30 degrees, and the magnetic beads move inward but can remain in the center and bottom of the reaction tube 13. Then, the magnetic beads 14 pass through the magnet in row 3, column 2 (3-2) of the outer magnetic block. Since the direction of the magnetic force generated by the magnet (3-2) is parallel to the horizontal plane, the angle between the direction of the magnetic force generated by the magnet (3-2) and the horizontal plane is 0 degrees, and the magnetic beads can move outward and upward to approach the horizontal plane. Similarly, the magnetic beads move upward, downward, inward, and outward together with the passing magnet. After the reaction tube 13 rotates once and returns to its original position, the magnetic beads 14 perform three reciprocating motions between the inside and outside, and one reciprocating motion between the upper and lower sides. Although the arrangement of the magnets in the horizontal rows and vertical columns in the magnetic block has been described above, those skilled in the art can adjust the arrangement of the magnets in the magnetic block as needed, including adjusting the number and positions of the horizontal rows and vertical columns, and are not limited thereby to the above description.

[0073] In the third or fourth embodiment, it is preferable to use a magnetic block that moves together with the reaction tube, and to place the electromagnet in the magnetic block 12. Therefore, this embodiment is effective in that the direction of the magnetic force can be changed as the centrifuge holder rotates and faces different directions by controlling the presence or absence of the magnetic force of the electromagnet.

[0074] Specific embodiment of the centrifugal reaction method 1) Remove 1 to 200 μL of the sample to be assayed (e.g., a cultured cell sample) from a sample chamber (e.g., a 1.5 mL centrifuge tube or a 96-well plate), and add 1 to 200 μL of the sample to be assayed to a reaction tube. 2) Remove 1 to 200 µL of lysis buffer from a reagent reservoir (e.g., a 1.5 mL centrifuge tube) and add 1 to 200 µL of lysis buffer to the reaction tube. 3) Run the centrifugation process at a low speed (e.g., under a centrifugal force of 0.05 to 10 g) to allow the specimen and lysis buffer to mix in the reaction area, thereby closing the check valve. 4) Remove 1 to 20 μL of magnetic beads from a reagent reservoir (e.g., a 1.5 mL centrifuge tube) and add 1 to 20 μL of magnetic beads to the reaction tube. 5) Remove 1 to 200 µL of binding buffer from the reagent reservoir (e.g., a 1.5 mL centrifuge tube) and add 1 to 200 µL of binding buffer to the reaction tube. 6) Run the centrifugation process at a low speed (e.g., under a centrifugal force of 0.05 to 10 g) so that the nucleic acids released from the specimen and the magnetic beads are mixed with the binding buffer in the reaction area, thereby allowing the check valve to close. 7) After the magnetic beads adsorbed to the magnetic block are dropped into the grooves of the reaction tube, a centrifugation process is carried out at high speed (e.g., under a centrifugal force of 100 to 500 g), the check valve is opened, and impurities and buffer in the mixture are discharged from the reaction area. 8) Remove 1 to 200 µL of wash buffer from a reagent reservoir (e.g., a 1.5 mL centrifuge tube) and add 1 to 200 µL of wash buffer to the reaction tube. 9) Run the centrifugation process at a low speed (e.g., centrifugal force 0.05-10 g) so that the nucleic acid-bound magnetic beads and the washing buffer are mixed in the reaction area, thereby allowing the check valve to close. 10) After the magnetic beads adsorbed to the magnetic block are dropped into the grooves of the reaction tube, a centrifugation process is carried out at high speed (e.g., under a centrifugal force of 100 to 500 g), the check valve is opened, and impurities and buffer in the mixture are discharged from the reaction area. 11) Remove 1 to 200 µL of elution buffer from the reagent reservoir (e.g., a 1.5 mL centrifuge tube) and add 1 to 200 µL of elution buffer to the reaction tube. 12) Run the centrifugation process at a low speed (e.g., under a centrifugal force of 0.05 to 10 g) so that the nucleic acid-bound magnetic beads and the elution buffer are mixed in the groove of the reaction tube, thereby keeping the check valve closed. 13) Allow the magnetic beads to be attracted to the magnetic block, thereby allowing the magnetic beads to collect in the reaction tube, positioned in close proximity to the centrifuge shaft, and fixed in place. 14) Measure the nucleic acid concentration using a spectrophotometer in the centrifugal reactor.

[0075] Therefore, the centrifugal reaction apparatus and the centrifugal reaction method of the present disclosure are suitable for carrying out molecular biological reactions. It is effective for performing a one-touch reaction. Under the influence of centrifugal force and magnetic force, the magnetic beads are driven and moved, and the reaction mixture can be thoroughly mixed in a short time. The centrifugal force and the reaction tube are used in molecular biological assay devices to save energy, save space, and greatly shorten the assay process. It helps to miniaturize the bioassay machine. Furthermore, the reaction process eliminates the need for laborious switching between different instruments to improve ease of use, ensure fast and safe operation, save energy, increase assay accuracy, achieve cost-effectiveness and prevent contamination.

[0076] The above-mentioned embodiments of the present disclosure are illustrative rather than limiting the present disclosure. Any equivalent modifications or changes made by those skilled in the art to the preferred embodiments without departing from the spirit of the present disclosure should be considered to be included in the claims of the present disclosure. [Explanation of symbols]

[0077] 1. Centrifugal reactor 11 Centrifugal Disc 111 Centrifugal shaft 112 Centrifuge holder 12 Magnetic Block 121 Inner magnetic block 122 Outer magnetic block 123 Upper magnetic block 124 Lower magnetic block 125 Side magnetic block 13 Reaction tube 14 Magnetic beads

Claims

1. A centrifugal reactor comprising: a centrifuge disk comprising a centrifuge shaft and at least one centrifuge holder, the centrifuge holder being arranged annularly around the centrifuge shaft; at least two magnetic blocks disposed on at least two sides of the centrifuge holder; Equipped with the centrifuge holder removably holds a reaction tube having at least one reaction region and at least one check valve, the reaction tube containing magnetic beads; the magnetic beads move in the reaction tube by receiving a sum of the magnetic forces of the at least two magnetic blocks and a centrifugal force; Centrifugal reactor.

2. the at least two magnetic blocks and the centrifugal disk are not connected and therefore do not move together; 2. The centrifugal reactor according to claim 1.

3. the at least two magnetic blocks are electromagnets, magnets, or a combination thereof; 3. The centrifugal reactor according to claim 2.

4. The at least two magnetic blocks and the centrifugal disk are connected and therefore move together; 2. The centrifugal reactor according to claim 1.

5. the at least two magnetic blocks are electromagnets; 5. The centrifugal reaction apparatus according to claim 4.

6. The at least two magnetic blocks and the centrifugal disk are spaced apart from each other by a distance of 0 to 300 mm.

2. The centrifugal reactor according to claim 1.

7. The centrifugal force is 1 to 80,000 g.

2. The centrifugal reactor according to claim 1.

8. the magnetic force of the at least two magnetic blocks is between 1 and 15,000 Gauss; 2. The centrifugal reactor according to claim 1.

9. The check valve of the reaction tube is electrically or magnetically controlled to open and close; The centrifugal reactor according to claim 8.

10. The magnetic beads are coated with biomolecules including antibodies, aptamers, peptides or nucleic acids.

2. The centrifugal reactor according to claim 1.

11. A centrifugal reaction method using the centrifugal reaction apparatus according to any one of claims 1 to 10, introducing at least one of a sample and / or a reaction reagent into the at least one reaction zone of the reaction tube in the centrifugal reaction device to form a reaction mixture; introducing magnetic beads into the at least one reaction area; performing a centrifugation process in the reaction tube under a first centrifugal force to initiate a first reaction, wherein the magnetic beads move in the reaction tube under a sum of the magnetic force of the at least two magnetic blocks and the centrifugal force; performing a centrifugation process on the reaction tube under a second centrifugal force to open the at least one check valve, thereby discharging a waste liquid of the first reaction, the second centrifugal force being greater than the first centrifugal force; The at least one check valve is disposed in a centrifugal direction of the at least one reaction region. method.

12. further comprising the step of introducing a wash buffer to remove impurities. The method of claim 11.

13. further comprising the step of recovering the product from the magnetic beads. The method of claim 11.

14. The magnetic beads are coated with biomolecules including antibodies, aptamers, peptides or nucleic acids. The method of claim 11.

15. The magnetic beads are coated with silicon dioxide. The method of claim 14.

16. the product is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); The method of claim 13.

17. A polymerase chain reaction is carried out in the at least one reaction area. The method of claim 11.

18. Nucleic acid hybridization is carried out in the at least one reaction area; The method of claim 11.

Images