WO2011150833A1

WO2011150833A1 - Test tube, device comprising test tube and liquid treatment method by using same

Info

Publication number: WO2011150833A1
Application number: PCT/CN2011/075017
Authority: WO
Inventors: 周宋兵; 居金良; 王敏; 朱慧伟; 黄鹤; 胡瑞
Original assignee: 上海仁度生物科技有限公司
Priority date: 2010-05-31
Filing date: 2011-05-31
Publication date: 2011-12-08
Also published as: CN101869855B; CN203244989U; CN101869855A; CN203030295U

Abstract

A test tube (10) used for liquid mixing and extracting operations is provided. The test tube (10) comprises an opening (12) for adding and withdrawing liquids, a near end portion (13) adjacent to the opening (12), and a far end portion (14) for mixing liquids and performing physicochemical reactions, wherein the near end portion (13) can be repeatedly switched between squeeze-to-shut state and loose-to-open state. A device for parallelly mixing and extracting a plurality of liquid samples is also provided. The device comprises the test tube (10). Furthermore, methods for mixing and extracting liquids by using the test tube (10) and the device are also provided. The test tube (10) and the device can conveniently and effectively overcome cross-contamination problem of liquids during mixing and extraction, and contribute to achieve automatic high flux operation of the liquid mixing and extraction processes. The test tube (10) and the device are particularly suitable to separate and detect target substances, e.g. nucleic acid, from biological samples.

Description

Test tube, device containing the same, and method of treating the same using the same

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test tube for liquid mixing and extraction operations, which is particularly suitable for separating and detecting a target substance such as a nucleic acid from a biological sample. The invention further relates to a device for mixing and extracting parallel operations of a plurality of liquid samples, said device comprising said test tubes. Further, the present invention provides a method of mixing and extracting a liquid using the test tube and apparatus.

BACKGROUND OF THE INVENTION In many fields of experimental science, such as chemistry, biology, medicine, and the like, liquid mixing and extraction processes are involved. For example, in the chemical field, a significant portion of the chemical reactions are carried out and completed in liquids. In the fields of biology and medicine, a large number of biological analysis tests and medical diagnosis require first to separate and purify labeled molecules from biological samples, such as separating characteristic proteins and nucleic acids from cells, and the separation and purification. The steps are also usually carried out in a liquid environment. In the various processes of liquid mixing and extraction, especially in the parallel operation of multiple samples, there are a series of continuous and cumbersome operation steps, such as adding liquid, pipetting, shaking mixing, adjusting physical and chemical parameters (such as temperature, pH), etc. A slight inadvertentness may cause cross-contamination of liquid samples, especially volatile liquids. Cross-contamination problems have therefore greatly increased the difficulty of achieving high throughput and automated control of liquid mixing and extraction processes. Take the nucleic acid separation and purification process as an example. Most nucleic acid isolation and purification methods include cell lysis, enzymatic treatment, separation of nucleic acids from other biomacromolecules, and purification of nucleic acids. Each step can be implemented individually or in combination by a number of different methods. The highly charged phosphate backbone of nucleic acids makes them more hydrophilic than other biomacromolecules such as proteins, polysaccharides, fats, etc. According to their differences in physicochemical properties, nucleic acids can be separated by selective precipitation, chromatography, density gradient centrifugation, etc. , purification. The main methods used in commercial applications are: centrifugation, membrane separation, nanoparticle-based magnetic beads, etc. (see, for example, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laborotary, etc., whose contents are complete Incorporated into this article). The typical feature of the centrifugation method is that it is separated by a centrifuge after high-temperature boiling, and nucleic acid and other macromolecular substances and other impurities are analyzed by taking the supernatant and removing the supernatant. The disadvantage is that it is difficult to automate operations. For example, the process of taking the supernatant and discarding the supernatant is difficult to implement in an automated manner. Another disadvantage is that the operation is cumbersome and time consuming. A skilled technician can only handle dozens of specimens a morning. Operators need to frequently operate equipment such as metals and centrifuges, and the operators are exposed to biological pollution throughout the process. The membrane separation method is represented by foreign companies such as AXYGEN and Qiagen, and is often used in scientific research and other fields. It is generally based on scientific research, with high cost and cumbersome operation.

Magnetic beads based on nanoparticles have been used more and more widely in recent years. In the magnetic bead method, after lysing cells by cell lysate, nucleic acid molecules released from the cells are specifically captured to the surface of the magnetic particles, and impurities such as proteins are not captured and remain in the solution. Subsequently, the magnetic particles are separated from the liquid under the action of a magnetic field, and the magnetic particles capturing the nucleic acid are recovered. Thereafter, the nucleic acid can be eluted from the magnetic particles with an eluent to obtain a purified nucleic acid. Since the magnetic bead method does not require centrifugation, does not require the addition of a plurality of reagents, is simple to operate, and meets the requirements for automated extraction of nucleic acids, and thus becomes an important development direction of nucleic acid purification methods. However, how to effectively overcome cross-contamination has always been an important issue to be solved in the high-throughput, automated operation of nucleic acid extraction. Due to the high sensitivity of the nucleic acid detection assay, any cross-contamination that occurs during the purification of the nucleic acid sample is likely to result in a positive result in subsequent assays. On the other hand, as described above, in a series of steps such as heating, shaking, loading, washing, etc., the trace liquid containing the nucleic acid is easily diffused into the air by means of an aerosol, causing cross-contamination between the samples. The currently known means of overcoming cross-contamination are mainly as follows:

1) The capping method, that is, the container containing the nucleic acid sample, such as a test tube, is sealed by using a cap, and the cap is opened only when liquid or liquid is required, thereby maximally avoiding the diffusion of the aerosol. However, the disadvantage of this method is that in the process of nucleic acid extraction and purification, it is necessary to repeatedly repeat the complicated operation of opening/closing the cover, which undoubtedly causes great obstacles for realizing the automatic operation of the machine;

2) The oil seal method, that is, the liquid surface of the biological sample containing the nucleic acid is sealed by using an oil layer to prevent the diffusion of the aerosol, and is added to the oil layer by a sample gun to perform liquid addition or liquid extraction. However, this method has limitations in the process of magnetic extraction of nucleic acids, because magnetic particles are transferred when the magnetic particles are transferred through the oil layer. The particles are easily adsorbed by the oil layer and remain in the oil layer, causing loss. Due to the above reasons, the current level of automation of nucleic acid extraction and purification process is still low. Only two companies, such as Gen-Probe and Roche, have FDA-approved automated nucleic acid extraction and detection systems. Therefore, in order to improve the high throughput and automation of nucleic acid extraction and purification, it is urgent to develop new, effective and convenient means to overcome cross-contamination. SUMMARY OF THE INVENTION The present invention is directed to a novel test tube and apparatus using the same, which facilitates and effectively overcomes the problem of cross-contamination of liquid during mixing and extraction, thereby overcoming the deficiencies of the prior art. The application of the new test tube can conveniently realize high-throughput, automated operation of the liquid mixing extraction process.

SUMMARY OF THE INVENTION In one aspect, the present invention provides a test tube that can be used for mixing and extracting liquids, the test tube including an opening for adding and aspirating liquid, a proximal portion adjacent to the opening, and for liquid mixing and physicochemical a distal portion of the reaction characterized in that: the proximal portion can be repeatedly replaced between a squeeze closed and a relaxed open state, which prevents liquid spillage present at the distal portion when squeezed closed or Evaporation, which allows the external pipette to extend or draw into the distal portion through the opening in the relaxed open state. In one embodiment, the proximal portion of the test tube is made of an elastic material. Preferably, the elastic material comprises silica gel. In another embodiment, the distal end portion of the test tube is made of a hard material, and the distal end portion includes a bottom portion and a side wall, wherein the distance and the angle between the bottom portion and the portion of the side wall are greater than The distance and angle of the side walls become a long bevel. In a further embodiment, the bevel forms a precipitation zone with the sidewall at the lower end. Preferably, the hard material comprises a PP polypropylene plastic. In certain embodiments, the proximal and distal portions of the test tube are two separate components during the production and dispensing phase, and the proximal and distal portions are assembled prior to the liquid mixing and extraction operation. The test tube of the present invention is integrally formed. For example, the proximal portion of the elastomeric material can be formed over the distal portion of the hard material to form a sealed interface prior to the liquid mixing and extraction operation, thus constituting the test tube of the present invention. In other embodiments, the proximal and distal portions of the test tube are integrally formed. In another aspect, the present invention provides a test tube for mixing and extracting liquids, the test tube comprising an opening for adding and aspirating liquid, a proximal portion adjacent to the opening, and for liquid mixing and physicochemical reaction The distal end portion is characterized in that: the distal end portion is made of a hard material, and the distal end portion includes a bottom portion and a side wall, wherein a distance and an angle between the bottom portion and a portion of the side wall portion are larger than the other portion side The distance and angle of the wall thus become a long bevel. Preferably, the hard material comprises a PP polypropylene plastic. In one embodiment, the bevel of the test tube forms a precipitation zone with the sidewall at the lower end. In a further embodiment, the proximal portion of the test tube can be repeatedly replaced between a squeeze closed and a relaxed open state, which prevents the presence of the distal portion when squeezed closed The liquid overflows or evaporates, which in the relaxed open state allows the outer straw to extend through the opening to the distal portion to add or aspirate the liquid. In still further embodiments, the proximal portion of the test tube is made of an elastomeric material, preferably comprising silica gel. In certain embodiments, the proximal portion and the distal portion of the test tube are sealingly joined. In other embodiments, the proximal and distal portions of the test tube are integrally formed. The liquids of the present invention can be various types of liquid mixtures including, but not limited to, various chemical substances or mixtures of biological materials and the like. In a preferred embodiment, the liquid of the invention comprises components derived from biological cells, including but not limited to proteins, nucleic acids, etc., preferably comprising nucleic acids. In still another aspect, the present invention provides a device for mixing and extracting parallel operations of a plurality of liquid samples, the device comprising a multi-position operating platform, each position of the operating platform accommodating one provided by the present invention a test tube containing an opening for adding and aspirating liquid, a proximal portion adjacent to the opening, and a distal portion for liquid mixing and physicochemical reaction, and the proximal portion can be repeatedly pressed and closed Relaxation between the two states, which prevents the liquid spillage or evaporation present in the distal portion when the squeeze is closed, allowing the outer straw to extend or extend into the distal portion through the opening in the relaxed open state Each position of the operating platform further includes a physical and chemical condition control component and a switch unit, wherein the physical and chemical condition control component contacts or is adjacent to a distal end portion of the test tube, and is capable of liquid sample of the distal end portion of The physicochemical conditions of the mixing and reaction are controlled, and the switching unit is in contact with or adjacent to the proximal end portion of the test tube, and the replacement control of the two states of the squeeze closing and the slack opening can be repeatedly performed on the proximal portion. Preferably, when the proximal end portion of the test tube is in the squeeze closed state, the test tube may be oscillated, mixed or heated to avoid liquid spillage or evaporation, while the proximal portion of the test tube is in a relaxed open state. , the test tube can be added or sucked up. In some embodiments, the physicochemical condition control includes, but is not limited to, control of one or more of a temperature, an oscillation frequency, and a magnetic field strength of the liquid of the distal portion, and the switch unit includes a Fixture. In one embodiment, the physical and chemical condition control unit and the switch unit of the plurality of positions may simultaneously perform parallel operations on a plurality of test tubes. In still further embodiments, the multi-position operating platform comprises a multi-well plate, each of the holes can accommodate a test tube provided by the present invention, and a plurality of the switch units are integrally constructed as another porous plate. The porous opening and closing on the plate can be controlled by an elastic device, and the porous portion of the plate can be repeatedly opened or closed at the same time by applying or withdrawing the force to the elastic device, thereby simultaneously pressing the proximal end portions of the plurality of test tubes. Close and slack open control for two states. In one embodiment, the apparatus provided by the present invention is used to separate a target component from a liquid sample. In a further embodiment, the liquid sample comprises a target component to be separated and magnetic particles capable of capturing the target component, and an outer wall of the distal end portion of the test tube is attached with a magnetic steel that can be evacuated, when When the magnetic steel is attached to the outer wall of the distal end portion, the magnetic particles are adsorbed, and when the magnetic steel is evacuated, the magnetic particles are scattered. Preferably, the distal end portion of the test tube is made of a hard material, and the distal end portion includes a bottom portion and a side wall, wherein a distance and an angle between the bottom portion and a portion of the side wall are greater than that of the other portion of the side wall The distance and the angle thus become a long bevel, and the magnetic steel that can be withdrawn is attached to the outer wall of the inclined surface. More preferably, the bevel forms a precipitation zone with the sidewall at the lower end. Optionally, the apparatus provided by the present invention may further comprise a detection device that can perform various required tests on the liquid sample, for example, performing various characterizations on the target component separated from the liquid sample, including However, it is not limited to determining the composition, structure, content, and the like of the target component. In one embodiment, the apparatus provided by the present invention operates in a fully automated manner. In still another aspect, the present invention provides a method of mixing and extracting a liquid sample, the method comprising: using the test tube provided by the present invention, the test tube comprising an opening for introducing and aspirating liquid, adjacent to the opening a proximal portion, and a distal portion for fluid mixing and physicochemical reaction, wherein the proximal portion of the tube can be repeatedly replaced between a squeeze closed and a relaxed open state, which is closed in compression The state prevents the liquid present in the distal portion from overflowing or evaporating, which allows the outer straw to extend or extend through the opening to the distal portion to add or aspirate the liquid. In one embodiment of the method of the present invention, when the liquid is withdrawn from or added to the test tube, the proximal portion of the test tube is in a relaxed open state, and at other times, the proximal end of the test tube The part is in a squeeze closed state. In another embodiment of the method of the present invention, the method comprises separating a target component from the liquid sample as follows: a) placing the proximal portion of the test tube in a relaxed state, The distal end portion of the test tube is added with a reagent for extracting a target component and a sample containing the target component, wherein the reagent for extracting the target component contains magnetic particles capable of capturing the target component; b) The proximal portion of the tube remains squeezed closed, the reagent and sample in the distal portion of the tube are mixed and allowed to undergo a physicochemical reaction, whereby the target component is captured by the magnetic particles; c) The outer wall of the distal end portion of the test tube is attached with magnetic steel that can be evacuated to adsorb the magnetic particles; d) the proximal portion of the test tube is again in a relaxed state, and the residual force is sucked from the test tube liquid. In a further embodiment of the method of the present invention, the method further comprises washing the separated target component by repeating the steps of: a) subjecting the proximal portion of the test tube to a relaxed opening a state of withdrawing the magnetic steel, adding a washing liquid; b) maintaining a proximal end portion of the test tube in a state of being squeezed closed, and shaking the test tube to cause the magnetic particles to be loosely dispersed in the washing liquid; c) reattaching the magnetic steel to the outer wall of the distal end portion of the test tube to adsorb the magnetic particles; and d) again causing the proximal portion of the test tube to be in a relaxed state, from The washing solution is aspirated from the test tube. In a preferred embodiment of the method of the invention, the distal end portion of the test tube is made of a hard material, and the distal end portion includes a bottom and a side wall, wherein the distance and the angle between the bottom portion and a portion of the side wall Greater than its distance from the other side walls and the angle to become a long bevel, the retractable magnet is attached to the outer wall of the bevel, and the bevel forms a precipitate at the lower end and the side wall a region, when the liquid is aspirated from the test tube, the outer straw extends into the precipitation zone for extraction, and when the liquid is added to the test tube, the outer straw extends toward the slope opposite the sedimentation zone The high end is used for force liquid. In a still further embodiment of the method of the present invention, the method further comprises detecting the separated target component. In a specific embodiment, the separated target component is retained in the distal portion of the test tube and the target component is directly detected. In another specific embodiment, the separated target component is transferred to another separate detection device to detect the target component. One skilled in the art will be able to select and use the various conventional detection methods known in the art to detect target components. For example, the protein component can be detected using an ELISA enzyme-linked immunoreaction (see, for example, Chinese Patent Application Publication No. CN1580772A, which is incorporated herein in its entirety). As another example, the nucleic acid component can be detected using chemiluminescence or real-time fluorimetry (see, e.g., U.S. Patent No. 7,169,554, U.S. Patent Application Publication No. US 2010/031152 A1, the entire disclosure of which is incorporated herein in its entirety). The method of mixing and extracting a liquid sample provided by the present invention can simultaneously perform a plurality of liquid samples in a parallel operation manner. Accordingly, the present invention also provides a parallel operation method for simultaneously mixing and extracting a plurality of liquid samples, the method comprising using the apparatus for mixing and extracting parallel operations of a plurality of liquid samples provided by the present invention, The device comprises a multi-position operating platform, each position of the operating platform can accommodate a test tube provided by the invention, and each position of the operating platform further comprises a physical and chemical condition control component and a switch unit, wherein the physical and chemical The condition control member contacts or is adjacent to the distal end portion of the test tube, and is capable of controlling the physical and chemical conditions of mixing and reaction of the liquid sample of the distal portion, and the switch unit contacts Or adjacent to the proximal end portion of the test tube, and the replacement control of the two states of the squeeze closing and the slack opening can be repeatedly performed on the proximal portion. Preferably, when the proximal end portion of the test tube is in the squeeze closed state, the test tube may be oscillated, mixed or heated to avoid liquid spillage or evaporation, while the proximal portion of the test tube is in a relaxed open state. , the test tube can be added or sucked up. In some embodiments, the physicochemical condition control comprises control of one or more of a temperature, an oscillation frequency, and a magnetic field strength of the liquid of the distal portion, and the switch unit includes a clamp. In one embodiment, the physical and chemical condition control unit and the switch unit can simultaneously perform parallel operations on the test tubes in a plurality of positions. In still further embodiments, the multi-position operating platform comprises a multi-well plate, each of the holes can accommodate a test tube provided by the present invention, and a plurality of the switch units are integrally constructed as another porous plate. The porous opening and closing on the plate can be controlled by an elastic device, and the porous on the plate can be repeatedly opened or closed at the same time by applying or withdrawing the force to the elastic device, thereby simultaneously pressing and closing the plurality of test tubes and relaxing the sheets. Open the replacement control for both states. The target component of the present invention may be, but is not limited to, a component derived from a biological cell including, but not limited to, a protein or a nucleic acid or the like, and is preferably a nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS For ease of understanding of the invention, the following figures are provided. In the following, specific embodiments of the invention will be described in conjunction with the drawings. However, it should be understood that the drawings and the specific embodiments are merely illustrative, and should not be construed as limiting the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view showing the structure of a specific embodiment of a test tube provided by the present invention. Fig. 2 is a schematic view showing the structure in which the proximal end portion of the test tube is pressed and closed by using a jig. 3a-3c are schematic views showing porous plates comprising a plurality of test tubes provided by the present invention from different angles. 4a-4b are partial schematic views of a multi-position operation platform provided by the present invention, wherein in FIG. 4a, The proximal end portion of the test tube is in a squeeze closed state, and in Fig. 4b, the proximal end portion of the test tube is in a relaxed open state. Figures 5 to 12 are schematic views showing separation, washing and transfer of a target component from a liquid sample, wherein:

Figure 5 shows the addition of a reagent for extracting a target component to a test tube in a state where the proximal portion of the test tube is in a relaxed state, the reagent containing magnetic particles capable of capturing the target component, and then being squeezed to close the test tube. The proximal portion; Figure 6 shows the addition of a sample containing the target component to the tube while the proximal portion of the tube is in a relaxed open state, and then squeezing the proximal portion of the tube closed; Figure 7 shows the tube in the tube While the proximal portion remains squeezed closed, the reagents and samples in the distal portion of the tube are mixed and their physicochemical conditions are adjusted, whereby the target component is captured by the magnetic particles; Figure 8 shows the holding of the proximal portion of the tube In the closed state, the magnetic particles are adsorbed on the inclined surface by the magnetic steel; Fig. 9 shows that the residual liquid is sucked from the sedimentation zone of the test tube while the proximal end portion of the test tube is in a relaxed state; Fig. 10 shows that the capture is performed. Washing of magnetic particles of the target component. Figure 11 shows the step in which the outer pipette is extended and mixed with the higher end of the slope opposite the sedimentation zone at the bottom of the tube. Figure 12 shows the step of transferring the target component from the precipitation zone at the bottom of the tube.

DETAILED DESCRIPTION OF THE INVENTION Referring to Figures 1 and 2, the present invention provides a test tube (10) that can be used for mixing and extracting liquids, the test tube containing an opening (12) for adding and aspirating liquid, proximal end adjacent the opening Part (13), and a distal portion (14) for liquid mixing and physicochemical reaction. As with conventional test tubes, the present invention provides a test tube having a cavity (11) in communication with the opening (12). Unlike other tubes known in the art, the proximal portion (13) of the test tube of the present invention can be repeatedly replaced between a squeeze closed and a relaxed open state, which is prevented in the squeeze closed state. Exist in the liquid outside the distal portion (14) Overflow or evaporation, which allows the outer straw to extend or extend through the opening to the distal portion (14) to add or aspirate the liquid. Repeated switching between the proximal end portion (13) of the test tube of the present invention in both the squeeze closed and relaxed open states can be accomplished using a variety of means. For example, the proximal portion (13) of the test tube may be a hose made of an elastic material, and the state of the hose is controlled by using an external clamp (20). When the clamp (20) clamps the clamping portion (29) on the hose, the hose is in a squeeze closed state, thereby closing the test tube; and when the clamp (20) is from the clamping portion on the hose ( 29) When released, the hose is in a relaxed open state, thereby opening the test tube. As another example, the proximal portion (13) of the test tube may be a hose made of an elastic material, and the state of the hose is controlled by using an external force. When no external force acts on the hose, the hose is naturally closed and closed. When an external force acts on the hose, the hose is pulled open, thereby opening the tube. The hose can be made using any elastic material known in the art as long as the elastic material is deformable in response to an external force and can be restored to its original state after the external force is removed. Preferably, an elastic material comprising silica gel can be used. The distal end portion (14) of the test tube of the present invention allows the liquid to mix therein and undergo a physicochemical reaction, and thus those skilled in the art can make any suitable shape using any suitable material as needed. A preferred embodiment is to use a hard material to form a rigid tube as shown in Figure 1. The hard tube has a bottom portion and a side wall, wherein a distance and an angle between the bottom portion and a portion of the side wall are greater than a distance and an angle between the bottom portion and the other side portions to form a long slope (15), the slope is lower One end and a side wall form a precipitation zone (16). The bottom shape with the bevel (15) and the precipitation zone (16) may be particularly advantageous in other processes for liquid mixing and extraction, particularly magnetic bead extraction of target components, such as nucleic acids, relative to other shapes. As shown in Figures 8 and 9, in the magnetic bead extraction process, the magnetic particles (24) can be adsorbed on the inclined surface (15) by the magnetic steel (25), and the external suction pipe (26) can easily reach the sediment at the bottom of the test tube. The region (16) exhausts the residual liquid and other substances, and easily avoids the magnetic particles (24), thereby effectively avoiding the aspiration of the magnetic particles (24). On the other hand, as shown in Fig. 11, when a liquid is added to the test tube, the outer straw extends to the higher end of the slope opposite to the sedimentation zone, whereby the magnetic particles (24) are added to the liquid While flushing into the precipitation zone (16) along the slope (15), it is sufficiently dispersed in the liquid so that the magnetic particles (24) are more easily mixed with the liquid. The rigid tube can be made using any hard material known in the art, preferably using a PP polypropylene material. The proximal end portion and the distal end portion of the test tube of the present invention may be separately fabricated, and then the partially dense portion Seal the connection. For example, a flexible hose can be made of a silicone material and a rigid tube made of PP polypropylene material, and then the elastic hose is placed over the hard tube to form a complete test tube. It is also possible to integrally form the proximal end portion and the distal end portion of the test tube of the present invention. Those skilled in the art will be well able to fabricate the test tubes of the present invention using conventional techniques in the art in light of the disclosure of this specification. As described above, due to the unique design of the test tube of the present invention, it is convenient to repeatedly switch the proximal end portion of the test tube between the two states of extrusion closing and slack opening, and thus is particularly suitable for performing a plurality of liquid samples. Mix and extract devices that operate in parallel. The present invention therefore also provides a device that can be used for mixing and extracting parallel operations of a plurality of liquid samples, the device comprising a multi-position operating platform, each of which can accommodate a test tube of the present invention. The multi-position operating platform can comprise a multi-well plate. For example, as shown in Figures 3a-3c, a plurality of test tubes provided by the present invention can be combined in a multiwell plate (17). The perforated plate (17) is provided with a plurality of through holes (18), and each of the through holes (18) is provided with a test tube (10) of the present invention. Optionally, a support (19) is attached below the perforated plate (17), which can better secure and support the test tube (10) of the present invention. Various porous plates can be used in the present invention, including but not limited to porous plates having 12 or fewer pores, 24 wells, 48 wells, 96 wells, 192 wells, 384 wells or more. In addition, each position of the multi-position operating platform further includes a switch unit that contacts or is adjacent to a proximal end portion of the test tube, and is capable of repeatedly pressing and closing the proximal portion Open the replacement control for both states. The switch unit may be a clamp (20) as shown in FIG. 2, when the clamp (20) clamps the proximal end portion of the test tube to be in a squeeze closed state, the test tube is closed; and when the clamp (20) The test tube is opened when the proximal portion is loosened to be in a relaxed open state. More preferably, the switch units of the plurality of positions can be constructed as one unit, and the plurality of test tubes are subjected to the replacement control of the two states of the squeeze closing and the slack opening. As an example, it is shown in Figure 4 that the switching units of the plurality of positions are integrally constructed into another plate comprising a porous body, the proximal end portion (13) of the test tube (10) passing through a hole in the plate, the opening of the hole The combination can be controlled by an elastic device. When an external force is applied to the elastic means by, for example, pushing the handle (30), the porosity on the flat plate can be simultaneously closed, so that the proximal end portion (13) of the plurality of test tubes (10) is in a squeeze-closed state, and when the force is removed, Simultaneously The perforation on the plate is opened such that the proximal portion (13) of the plurality of tubes (10) is in a relaxed open state. Alternatively, an external force may be applied to the elastic device by pulling the handle (30) to simultaneously open the perforation on the plate such that the proximal portion (13) of the plurality of test tubes (10) is in a relaxed open state, and when the force is removed, At the same time, the porosity on the plate is closed so that the proximal end portion (13) of the plurality of test tubes (10) is in a squeeze closed state. A variety of conventional elastic means capable of opening and closing the holes are known to those skilled in the art and can be applied to the switch unit of the present invention. Each position of the operating platform of the present invention further includes a physical and chemical condition control member that contacts or is adjacent to the distal end portion of the test tube, thereby enabling physical and chemical conditions for mixing and reacting the liquid sample of the distal portion. control. Those skilled in the art can design and set different physical and chemical control components according to the needs of a specific process. For example, by setting an oscillator to achieve thorough mixing of the liquid in the test tube, by adjusting a temperature setting device such as a water bath to adjust the temperature of the liquid located in the test tube, or by using a magnetic steel at the bottom of the test tube to change the magnetic field at the distal end portion of the test tube. Wait. In some embodiments, the parallel operation of the physical and chemical control components at multiple locations can be accomplished by conventional techniques in the art to simultaneously perform the same adjustments to the physical and chemical conditions of the liquid in the plurality of test tubes. Optionally, the apparatus provided by the present invention may further comprise a detection device that can perform various desired tests on the liquid sample. For example, when the device provided by the present invention is used to separate a target component such as a protein or a nucleic acid derived from a biological cell from a biological sample, the detecting device can perform various characterizations on the separated target component, including However, it is not limited to determining the composition, structure, content, and the like of the target component. In one embodiment, the detection device is configured to contact or be adjacent to the distal portion of the tube so that it can be directly detected without transferring the sample within the tube. In another embodiment, the detection device is included as a stand-alone module in a device provided by the present invention, and the sample is detected by transferring a liquid sample from the test tube to the test device. Further, the present invention provides a method of mixing and extracting liquids using a test tube provided by the present invention. Preferably, in the method provided by the present invention, the proximal end portion of the test tube provided by the present invention is in a relaxed open state to open the test tube only when liquid is added to or taken from the distal end portion of the test tube, and at other times For example, when heating, oscillating, and standing, the proximal end portion of the test tube provided by the present invention is in a squeeze closed state thereby closing the test tube, thereby maximally reducing the external contamination of the sample, especially the cross-contamination between the samples. chance. As described above, the method of the present invention is applicable to various needs for mixing, extracting or generating liquids. The process of physicochemical reaction. Particularly preferably, the method of the invention is suitable for isolating a target component from a biological sample, including but not limited to a protein or nucleic acid, from a biological sample. Particularly preferably, the target component is a nucleic acid. Therefore, the method of the present invention will be described below by taking a nucleic acid separation and purification process as an example. However, those skilled in the art will appreciate that the method of the present invention can be equally applied to mixing or extraction processes for any other liquid as desired. By way of example, Figures 5-10 provide a process for isolating nucleic acids from a biological sample comprising the steps of: as shown in Figure 5, when the proximal portion (13) of the tube (10) is in a relaxed state, An external pipette (23) adds a nucleic acid extract (21) to the test tube (10), the nucleic acid extract (21) containing magnetic particles capable of specifically capturing a target nucleic acid, and then placing the proximal portion (13) Squeeze off. Various methods of specifically capturing a target nucleic acid are known in the present invention. For example, an oligonucleotide strand capable of achieving specific hybridization with a specific sequence on a target nucleic acid can be covalently attached to the surface of the magnetic particle, and the target nucleic acid can be captured by hybridization between the oligonucleotide strand and the target nucleic acid. Magnetic particle surface. Subsequently, as shown in Fig. 6, the proximal portion (13) of the test tube (10) is again brought into a relaxed state, and an external pipette is used to add a sample (22) to the test tube (10) containing the target to be separated. Nucleic acid. After the loading is completed, the proximal portion (13) is again brought into a squeeze closed state. It should be understood that the nucleic acid extract and sample may be added in any order or simultaneously. Thereafter, as shown in Fig. 7, the proximal end portion (13) of the test tube (10) is kept in a squeeze-closed state, and the test tube is shaken to sufficiently mix the nucleic acid extract (21) with the sample (22). Subsequently, the distal portion (14) of the test tube (10) is heated to lyse the biological cells in the sample to release the nucleic acid. For example, the sample can be lysed by heating the sample to 60 ° C in a water bath for 8 minutes. After the cleavage is completed, the magnetic particles capture the nucleic acid at room temperature (e.g., 25 ° C). In the next step, as shown in Fig. 8, the proximal end portion (13) of the test tube (10) is kept in a squeeze-closed state, and the magnetic steel (25) is attached to the outer wall of the test tube (10) to adsorb the test tube (10). Magnetic particles (24) inside. Preferably, the magnetic steel (25) is attached to the bottom bevel (15) of the test tube (10). The magnetic particles (24) may be ferrite materials, and the magnetic steel (25) may be natural magnets. Subsequently, the proximal portion (13) of the test tube (10) is left in a relaxed state, using an external straw (26) The raffinate is withdrawn from the test tube (10), preferably from the precipitation zone (16) at the bottom of the test tube (10). Through the above steps, the target nucleic acid is captured on the magnetic particles, and sufficiently separated from the nucleic acid reaction solution and other components in the sample, thereby obtaining a purified target nucleic acid. The obtained target nucleic acid can be further washed as needed. As shown in Fig. 10, the magnetic steel (25) is withdrawn, and the proximal portion (13) of the test tube (10) is in a relaxed open state, and a washing liquid (27) is added to the test tube (10); 10) The proximal portion (13) is in a squeeze-closed state, and after oscillating and mixing the washing liquid (27) and the magnetic particles (24), the magnetic steel (25) is again attached to the outer wall of the test tube (10) such as a test tube ( 10) on the bottom slope (15) to adsorb the magnetic particles (24) in the test tube (10); finally, the proximal portion (13) of the test tube (10) is in a relaxed open state, using an external straw (26) The test tube (10), preferably the wash solution is aspirated from the precipitation zone (16) at the bottom of the test tube (10). The washing step can be repeated as many times as needed. The purified nucleic acid or protein can be subjected to further manipulation, for example, detection of the isolated nucleic acid or protein for the purpose of isolating and purifying the nucleic acid or protein. A variety of methods for detecting nucleic acids or proteins are known in the art, including, but not limited to, chemiluminescence methods (see, for example, U.S. Patent No. 7,169,554, the entire disclosure of which is incorporated herein in its entirety), Application Publication No. US2010/031152A1, which discloses a method and system for quantifying nucleic acids by real-time nucleic acid amplification, which is incorporated herein in its entirety, as well as various antigen-antibody-based assays (eg, ELISA, See, for example, Chinese Patent Application Publication No. CN1580772A, the entire disclosure of which is incorporated herein in its entirety in its entirety in its entirety in the the the the the the The detection can be carried out directly in a test tube. For example, various reagents required for detection may be added to the distal portion of the test tube (10), and a detection device such as a fluorescence excitation device and a reading device, or a spectrophotometer may be disposed outside the distal portion. The entire process of extraction, purification, and detection can be completed without transferring the nucleic acid. The detection may also be carried out by transferring the purified nucleic acid to another detection device, such as a micro-reaction plate (28), which may be, but is not limited to, a 96-well plate, as shown in FIG. The processing steps for a single liquid sample are described above, taking only a single tube as an example. It is well understood by those skilled in the art that by using the apparatus for mixing and extracting parallel operations of a plurality of liquid samples provided by the present invention, multiple liquid samples of a plurality of test tubes can be simultaneously operated in parallel. Therefore, the present invention also covers a parallel operation of simultaneously mixing and extracting a plurality of liquid samples. Law. As described above, by the parallel operation of the physical and chemical condition control unit and the switch unit of each position of the multi-position operation platform included in the apparatus of the present invention, the open or closed state of the plurality of test tubes can be simultaneously controlled in parallel, and The physical and chemical conditions of the liquid sample are controlled by synchronization.

It is to be understood that the embodiments and the specific embodiments of the present invention are intended to be illustrative and not restrictive. Various modifications, substitutions, omissions and additions may be made by those skilled in the art without departing from the spirit of the invention. The scope of the invention is to be limited only by the claims and the equivalents thereof.

Claims

Claim

A test tube for mixing and extracting a liquid, the test tube comprising an opening for adding and extracting a liquid, a proximal portion adjacent to the opening, and a distal portion for mixing and physicochemically reacting the liquid, The feature is that the proximal portion can be repeatedly replaced between a squeeze closed state and a relaxed open state, which prevents the liquid present in the distal end portion from overflowing or evaporating in the squeeze closed state, which is loosened open The state allows the external pipette to extend or extend through the opening to the distal portion to add or aspirate the liquid.

The test tube according to claim 1, wherein: the proximal end portion of the test tube is made of an elastic material.

3. The test tube according to claim 2, wherein: said elastic material comprises silica gel.

4. The test tube of claim 1 wherein said distal end portion is made of a hard material and said distal end portion includes a bottom portion and a side wall, wherein said bottom portion is spaced from said portion of said side wall and The angle is larger than the distance and angle between the other side walls and becomes a long slope.

The test tube according to claim 4, wherein: said slope forms a sedimentation zone at the lower end with the side wall.

6. A test tube according to claim 4 or 5, wherein: said hard material comprises PP polypropylene plastic.

7. A test tube for mixing and extracting a liquid, the test tube comprising an opening for adding and extracting a liquid, a proximal portion adjacent to the opening, and a distal portion for mixing and physicochemically reacting the liquid, The feature is: the distal end portion is made of a hard material, and the distal end portion includes a bottom portion and a side wall, wherein the distance and the angle between the bottom portion and the partial side wall are greater than the distance and angle between the bottom portion and the other portion of the side wall Thereby becoming a long bevel.

The test tube according to claim 7, wherein: said slope forms a sedimentation zone with the side wall at a lower end.

9. A test tube according to claim 7 or 8, wherein: said hard material comprises PP polypropylene plastic.

10. The test tube of claim 7 wherein: said proximal portion of said test tube is Replacing between being repeatedly pressed and relaxed, which prevents liquid spillage or evaporation existing in the distal portion when in the squeeze closed state, which allows in the relaxed state An outer straw extends through the opening to the distal portion to add or aspirate liquid.

A test tube according to claim 10, wherein: said proximal end portion of said test tube is made of an elastic material.

12. The test tube of claim 11 wherein: said elastomeric material comprises silica gel.

13. A test tube as claimed in claim 1 or claim 7 wherein: said liquid comprises a component derived from a fine material of a bio-pack.

The test tube according to claim 13, wherein: said component derived from biological cells is selected from a protein or a nucleic acid.

The test tube according to any one of claims 1 to 12, wherein: the proximal end portion and the distal end portion are sealingly connected.

The test tube according to any one of claims 1 to 12, wherein the proximal end portion and the distal end portion are integrally formed.

17. A device for mixing and extracting parallel operations of a plurality of liquid samples, the device comprising a multi-position operating platform, each of the operating platforms being capable of accommodating one of claims 1-6 The test tube, each position of the operating platform further comprises a physical and chemical condition control component and a switch unit, wherein the physical and chemical condition control component contacts or is adjacent to a distal end portion of the test tube, and is capable of facing the distal end The mixing of the partial liquid sample and the physicochemical conditions of the reaction are controlled, and the switch unit contacts or is adjacent to the proximal end portion of the test tube, and can repeatedly perform the squeeze closing and the slack opening on the proximal end portion. Replacement control of the state.

18. Apparatus according to claim 17 wherein: said physical and chemical condition control comprises control of one or more of temperature, oscillation frequency and magnetic field strength of said distal portion.

19. Apparatus according to claim 17 wherein: said switch unit is a clamp.

20. The apparatus according to claim 17, wherein: when the proximal end portion of the test tube is in a squeeze-closed state, the test tube can be oscillated, mixed or heated to avoid liquid spillage or evaporation, and When the proximal part of the test tube is in a relaxed state, the test tube can be added Or the operation of drawing liquid.

The apparatus according to claim 17, wherein: physicochemical condition control means and switching means of said plurality of positions are capable of simultaneously performing parallel operations on a plurality of test tubes.

22. The apparatus of claim 17 wherein: said multi-position operating platform comprises a multi-well plate, each of said holes accommodating a test tube according to any of claims 1-6, and The switch unit is integrally constructed as another porous plate, and the porous opening and closing on the plate can be controlled by an elastic device, and the porous on the plate can be repeatedly opened by applying or withdrawing the force to the elastic device. Or closed, so that the proximal end portions of the plurality of test tubes are subjected to the replacement control of the two states of the squeeze closing and the slack opening.

23. Apparatus according to claim 17 wherein: said liquid sample comprises a target component to be separated and magnetic particles capable of capturing said target component.

The apparatus according to claim 23, wherein: the outer wall of the distal end portion of the test tube is attached with a magnetic steel which can be evacuated, and when the magnetic steel is attached to the outer wall of the distal end portion The magnetic particles are adsorbed, and when the magnetic steel is evacuated, the magnetic particles are dispersed.

25. Apparatus according to claim 24 wherein: said distal end portion of said test tube is made of a hard material, and said distal end portion includes a bottom portion and a side wall, wherein said bottom portion and a portion of said side wall portion The distance and the angle are larger than the distance and angle from the other side walls to form a long bevel, and the magnetic steel which can be detached is attached to the outer wall of the inclined surface.

26. Apparatus according to claim 25 wherein: said ramp forms a settling zone with the side walls at a lower end.

27. Apparatus according to claim 23 wherein: said target component is a component from a biological packet.

28. Apparatus according to claim 27 wherein: said components from biological cells are selected from proteins or nucleic acids.

29. Apparatus according to claim 28 wherein: said component from the biological cell is a nucleic acid.

30. Apparatus according to any of claims 17-29, wherein: said apparatus is Run in automatic mode.

A method of mixing and extracting a liquid sample, the method comprising: using the test tube according to any one of claims 1-6.

32. The method according to claim 31, wherein: when the liquid is withdrawn from or added to the test tube, the proximal end portion of the test tube is in a relaxed state, and at other times, The proximal portion of the tube is in a squeeze closed state.

33. The method of claim 31, wherein: the method comprises separating the target component from the liquid sample as follows: a) causing the proximal portion of the test tube to be in a relaxed state And a reagent for extracting a target component and a sample containing the target component are added to the distal end portion of the test tube, wherein the reagent for extracting the target component contains magnetic particles capable of capturing the target component; b) maintaining the proximal portion of the test tube in a squeeze-closed state, mixing the reagent and the sample at the distal end portion of the test tube and allowing it to undergo a physical and chemical reaction, whereby the target component is captured by the magnetic particles; c) attaching a magnetic steel that can be evacuated to the outer wall of the distal end portion of the test tube to adsorb the magnetic particles; d) again bringing the proximal end portion of the test tube to a relaxed state, and from the test tube Aspirate the residual liquid.

34. The method of claim 33, wherein: the method further comprises: washing the separated target component by repeating the steps of: a) placing the proximal portion of the test tube at a state of relaxation and opening, evacuating the magnetic steel, adding a washing liquid; b) maintaining the proximal end portion of the test tube in a state of being squeezed closed, and shaking the test tube to cause the magnetic particles to be loosely dispersed in the washing liquid; Placing the magnetic steel again on the outer wall of the distal end portion of the test tube to adsorb the magnetic particles; d) The proximal portion of the test tube is again brought into a relaxed state, and the washing liquid is aspirated from the test tube.

35. The method of claim 33 or 34 wherein: the target component is a component from a biological fines.

36. The method of claim 35, wherein: the component from the biological cell is selected from the group consisting of a protein or a nucleic acid.

37. The method of claim 36 wherein: said component from the biological cell is a nucleic acid.

38. The method of claim 33 or 34, wherein: the distal end portion of the test tube is made of a hard material, and the distal end portion includes a bottom portion and a side wall, wherein the bottom portion and a portion of the side portion The distance and angle of the wall are greater than the distance and angle between the wall and the other side walls to form a long bevel, the retractable magnetic steel is attached to the outer wall of the inclined surface, and the inclined surface is lower One end forms a sedimentation zone with the side wall, and when the liquid is sucked from the test tube, the outer pipette extends into the sedimentation zone for extraction, and when the liquid is added to the test tube, the outer pipette extends to the sedimentation zone. The opposite end of the inclined surface is filled with liquid.

39. The method of any of claims 31-38, wherein: the method comprises parallel operation of simultaneously mixing and extracting a plurality of liquid samples, the method comprising using one for multiple a liquid sample for mixing and extracting a device for parallel operation, the device comprising a multi-position operating platform, each position of the operating platform accommodating a test tube according to any one of claims 1-6, said operation Each position of the platform further includes a physical and chemical condition control unit and a switch unit, wherein the physical and chemical condition control unit contacts or is adjacent to the distal end portion of the test tube, and is capable of mixing and reacting the liquid sample of the distal portion The physicochemical conditions are controlled, and the switch unit contacts or is adjacent to the proximal end portion of the test tube, and is capable of repeatedly performing the replacement control of the squeeze close and slack open states for the proximal portion.

40. The method of claim 39, wherein: said physical and chemical condition control comprises control of one or more of a temperature, an oscillation frequency, and a magnetic field strength of said distal portion.

41. The method of claim 39, wherein: said switch unit is a clamp.

42. The method according to claim 39, wherein: when the proximal end portion of the test tube is in a squeeze closed state, the test tube can be oscillated, mixed or heated to avoid liquid spillage or evaporation, and When the proximal portion of the test tube is in a relaxed open state, the test tube may be subjected to an operation of adding or sucking a liquid.

43. The method according to claim 39, wherein: said physical and chemical condition control unit and said switching unit are capable of simultaneously performing parallel operations on the test tubes in the plurality of positions.

44. The method of claim 39, wherein: said multi-position operating platform comprises a multi-well plate, each of said holes accommodating a test tube according to any of claims 1-6, and The switch unit is integrally constructed as another porous plate, and the porous opening and closing on the plate can be controlled by an elastic device, and the porous on the plate can be repeatedly opened by applying or withdrawing the force to the elastic device. Or close, so that the multiple tubes are simultaneously subjected to the compression control of the two states of the squeeze closing and the slack opening.