WO2020248203A1 - 离心式反应微管、离心式反应装置及其离心式检验方法 - Google Patents

离心式反应微管、离心式反应装置及其离心式检验方法 Download PDF

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Publication number
WO2020248203A1
WO2020248203A1 PCT/CN2019/091171 CN2019091171W WO2020248203A1 WO 2020248203 A1 WO2020248203 A1 WO 2020248203A1 CN 2019091171 W CN2019091171 W CN 2019091171W WO 2020248203 A1 WO2020248203 A1 WO 2020248203A1
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Prior art keywords
reaction
centrifugal
microtube
way valve
reaction zone
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PCT/CN2019/091171
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English (en)
French (fr)
Inventor
王锦弘
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王锦弘
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Application filed by 王锦弘 filed Critical 王锦弘
Priority to JP2021573739A priority Critical patent/JP7390402B2/ja
Priority to CN201980096945.9A priority patent/CN114173924A/zh
Priority to PCT/CN2019/091171 priority patent/WO2020248203A1/zh
Priority to US17/616,104 priority patent/US20220314214A1/en
Priority to EP19932490.6A priority patent/EP3984638A4/en
Publication of WO2020248203A1 publication Critical patent/WO2020248203A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0621—Control of the sequence of chambers filled or emptied
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0605—Valves, specific forms thereof check valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated

Definitions

  • the application relates to a reaction microtube and a reaction device for experiments, in particular to a reaction microtube and a reaction device that complete molecular biological detection through one-click.
  • PCR polymerase chain reaction
  • the purpose of the present invention is to provide a centrifugal reaction microtube and a reaction device.
  • extraction, reaction, cleaning and cleaning can be performed in the same reaction microtube by centrifugation. / Or detect the signal to achieve the effect of reducing manpower demand and pollution.
  • an embodiment of the present invention provides a centrifugal reaction microtube, including:
  • the first reaction zone is used to contain the reaction mixture for the first reaction.
  • the first check valve is located in the centrifugal direction of the first reaction zone, wherein the first check valve is opened by the first centrifugal force to allow the reaction mixture to leave the first reaction zone.
  • the centrifugal reaction microtube further includes:
  • the second reaction zone is separated from the first reaction zone by the first one-way valve, and the reaction mixture is moved to the second reaction zone by the first centrifugal force to perform the second reaction;
  • the second one-way valve is arranged on the opposite side of the first one-way valve in the second reaction zone, wherein the second one-way valve is opened by means of a second centrifugal force to allow the reaction mixture to leave the second reaction zone.
  • the second centrifugal force is greater than the first centrifugal force.
  • the first centrifugal force and the second centrifugal force may be 1 to 80,000 g, respectively, and the second centrifugal force is greater than the first centrifugal force.
  • the opening and closing of the first one-way valve and the second one-way valve can be controlled by electronic control or magnetic control.
  • the centrifugal reaction microtube may further comprise a biochip arranged in the second reaction zone, on which biomolecules including antibodies, aptamers, peptides or nucleic acids are coated.
  • the centrifugal reaction microtube may further include a waste liquid area, separated from the second reaction area by means of a second one-way valve.
  • the first reaction zone can perform a polymerase chain reaction and/or the second reaction zone can perform a nucleic acid hybridization reaction.
  • the centrifugal reaction microtube further includes:
  • the third reaction zone is used to contain the reaction mixture for the third reaction.
  • the third check valve is arranged between the third reaction zone and the first reaction zone, and the reaction mixture is moved to the first reaction zone by means of the third centrifugal force.
  • the centrifugal reaction microtube can be substantially made of optical materials. More preferably, the optical material may include quartz, glass or plastic.
  • an embodiment of the present invention provides a centrifugal reaction device, including:
  • the reaction centrifugal disc is inclined upwardly from the shaft center, and the reaction centrifugal disc is provided with a plurality of fixed racks symmetrically arranged for accommodating a plurality of centrifugal reaction microtubes.
  • the slope may be about 1 to 89 degrees.
  • the centrifugal reaction device may further include a signal detection module for detecting the reaction signal of the centrifugal reaction microtube.
  • the centrifugal reaction device may further include a liquid separation module to add reaction reagents and/or samples into the centrifugal reaction microtubes.
  • the centrifugal reaction device may further include a temperature control module for controlling the reaction temperature of the centrifugal reaction microtube.
  • the centrifugal reaction device may further include a waste liquid collection module for collecting waste liquid leaving from the centrifugal reaction microtube.
  • an embodiment of the present invention provides a centrifugal detection method, including:
  • the sample and/or reaction reagent are added to the first reaction zone to form a reaction mixture, and the centrifugal reaction microtube is centrifuged to perform the first reaction.
  • the method may further include adding a cleaning solution and intermittently centrifuging the centrifugal reaction microtube.
  • the method may further include detecting the reaction signal of the centrifugal reaction microtube.
  • the method may further include controlling the reaction temperature of the centrifugal reaction microtube.
  • the method may further include collecting waste liquid exiting from the centrifugal reaction microtube.
  • the method may further include:
  • the centrifugal reaction microtube is used to move the reaction mixture through the first one-way valve to the second reaction zone for the second reaction by means of the first centrifugal force.
  • the method may further include:
  • the centrifugal reaction microtube is centrifuged, and the reaction mixture leaves the second reaction zone through the second one-way valve by means of the second centrifugal force.
  • the second centrifugal force is greater than the first centrifugal force.
  • the first centrifugal force and the second centrifugal force may be 1 to 80,000 g, respectively, and the second centrifugal force is greater than the first centrifugal force.
  • the opening and closing of the first one-way valve and the second one-way valve can be further controlled by electronic control or magnetic control.
  • the first reaction zone can perform a polymerase chain reaction and/or the second reaction zone can perform a nucleic acid hybridization reaction.
  • Fig. 1 is a schematic diagram of a centrifugal reaction microtube according to the first embodiment of the present application.
  • Fig. 2 is a schematic diagram of a centrifugal reaction microtube according to a second embodiment of the present application.
  • Fig. 3 is a schematic diagram of a centrifugal reaction microtube according to a third embodiment of the present application.
  • FIG. 4(A)-FIG. 4(C) are schematic diagrams of the use state of the centrifugal reaction microtube according to the 3A embodiment of the present application.
  • 5(A) and 5(B) are schematic diagrams of the centrifugal reaction microtubes according to the fourth and 4A embodiments of the present application, respectively.
  • Fig. 6 is a schematic diagram of a centrifugal reaction microtube according to a fifth embodiment of the present application.
  • Fig. 7 is a schematic diagram of a centrifugal reaction microtube according to a sixth embodiment of the present application.
  • Fig. 8 is a schematic diagram of a centrifugal reaction microtube according to a preferred embodiment of the present application.
  • Fig. 9 is a schematic diagram of a reaction centrifuge disk according to an embodiment of the present application.
  • a centrifugal reaction microtube which includes: at least a reaction zone for containing a reaction mixture including a reaction reagent, a cleaning solution and/or a sample for reaction; and at least one-way valves, which are respectively located in the reaction zone.
  • the reaction mixture flows away from the axis by means of centrifugal force, and when the centrifugal speed or centrifugal force exceeds the threshold, the check valve is opened to make the reaction mixture leave the reaction zone, and when the centrifugal speed or centrifugal force is lower than the threshold
  • the one-way valve is closed to prevent the reaction mixture from returning to the original reaction zone.
  • the reaction microtubule can be divided into at least one reaction zone for the purification, amplification and analysis of analytes (such as proteins, nucleic acids and other biological molecules) .
  • analytes such as proteins, nucleic acids and other biological molecules
  • the "one-way valve” means that the reaction reagent, cleaning solution and/or sample can be controlled to remain in the reaction zone when not centrifuged, and the reaction reagent, cleaning solution and/or sample can be controlled during centrifugation. A mechanism that passes through the "one-way valve" to another reaction zone without backflow.
  • a mechanical check valve is composed of springs with different elastic constants and balls with different weights.
  • the material can be metal or non-metal. Different sizes of centrifugal force can correspond to valve bodies with different elastic constants, allowing different valve bodies in the reaction microtube to open or close under different sizes of centrifugal force. That is, with the use of centrifugal force, the one-way valve can control the retention or elimination of the reaction mixture.
  • the one-way valve can also be opened and closed by electronic control or magnetic control.
  • the compression spring is fully extended and the one-way valve is completely sealed under the condition that there is no centrifugal force or the compression stress of the spring.
  • the centrifugal force reaches the compression stress of the spring, as the centrifugal force gradually increases, the compression spring will gradually become shorter due to compression, thereby opening the one-way valve so that the reaction mixture can move through the one-way valve in the direction away from the rotation axis.
  • the waste liquid is collected in an airtight manner, that is, the centrifugal reaction microtube may further include a waste liquid zone, which is separated from the reaction zone by means of a one-way valve, so that the reaction mixture or cleaning liquid after the reaction in the reaction zone is separated by After centrifugation, it can leave the reaction zone to the waste liquid zone without refluxing.
  • a biochip may be provided in the reaction zone of the centrifugal reaction microtube, on which biomolecules are coated for hybridization with the reaction mixture.
  • Biomolecules may include antibodies, aptamers, peptides or nucleic acids, and the like.
  • the centrifugal reaction microtube 1 includes a reaction zone 11 and a one-way valve 12.
  • the nucleic acid molecules amplified by PCR can react with the biochip pre-coated with target nucleic acid in the reaction zone 11, and the partially complementary nucleic acid molecules will be attached to the biochip, and then centrifuged The unattached nucleic acid molecules are removed through the one-way valve 12.
  • the centrifugal reaction microtube 2 includes a reaction zone 11 and a one-way valve 12, and further includes a waste liquid zone 13, which can be used to collect the waste liquid after the reaction.
  • the centrifugal reaction microtube 3 includes a first reaction zone 111, a first check valve 121, a second reaction zone 112 and a second check valve 122.
  • the first reaction zone 111 can be used for the first reaction of the reaction mixture;
  • the first one-way valve 121 is located in the centrifugal direction of the first reaction zone 111;
  • the second reaction zone 112 is separated from the first reaction zone 111 by the first one-way valve 121;
  • the second one-way valve 122 is arranged on the opposite side of the first one-way valve 121 in the second reaction zone 112.
  • the polymerase chain reaction can be carried out in the first reaction zone 111 and/or nucleic acid hybridization reaction can be carried out in the second reaction zone 112; or nucleic acid extraction can be carried out in the first reaction zone 111 and/or nucleic acid can be carried out in the second reaction zone 112 Hybridization reaction.
  • those skilled in the art can select springs with different or the same elastic constants according to requirements to control the opening and closing of the first one-way valve 121 and the second one-way valve 122 under the same centrifugal conditions or under the same centrifugal conditions. Open and close under different centrifugal conditions.
  • balls of different weights can be used, that is, a heavier ball is used for the first one-way valve 121, and a heavy ball is used for the second one-way valve 122. Since the ball is lighter, the centrifugal force required to open the first one-way valve 121 is smaller than the centrifugal force required to open the second one-way valve 122.
  • the elastic constant of the spring of the second one-way valve 122 is greater than the elastic constant of the spring of the first one-way valve 121.
  • the first centrifugal force and the second centrifugal force may be 1 to 80,000 g, respectively, and the second centrifugal force is greater than the first centrifugal force.
  • the centrifugal reaction microtube includes a first reaction zone 111, a first check valve 121, a second reaction zone 112 and a second check valve 122.
  • the difference from the third embodiment is that the first check valve 121 and the second check valve 122 are controlled by a single spring.
  • the first check valve 121, the second reaction zone 112, and the second check valve 122 jointly form a sliding member, which can slide in the direction of centrifugal force during centrifugation.
  • the sliding member can be connected with the spring, so that the sliding member can return to the static state after centrifugation.
  • the spring may be a compression spring, which is arranged on the right side of the sliding member in the figure; or the spring may be a tension spring, which is arranged on the left side of the sliding member.
  • FIG. 4(A) shows an unprocessed centrifugal reaction microtube, the first reaction zone 111 and the second reaction zone 112 are not connected, and the slider is in the first position of the static state.
  • the sliding member moves to the second position shown in (B), so that the through hole is connected to the first check valve 121, and the reaction reagent, cleaning solution and/or sample will be removed from the first check valve 121.
  • a reaction zone 111 passes through the first one-way valve 121 to the second reaction zone 112, but the second one-way valve 122 has not yet communicated with the through hole, so the reaction reagent, cleaning solution and/or sample will not pass through the second one-way valve 122.
  • the sliding member When the second centrifugal force is greater than the first centrifugal force for centrifugation, the sliding member further moves to the third position shown in (C), so that the through hole is communicated with the second check valve 122, and the through hole is not connected to the first one.
  • the one-way valve 121 is in communication, so the reaction reagent, the cleaning solution and/or the sample will be removed from the second reaction zone 112 through the second one-way valve 122, and will not flow back to the first reaction zone 111.
  • the centrifugal reaction microtube 4 includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, and a second check valve 122. , Further includes a waste liquid area 13, which can be used to collect the waste liquid after the reaction.
  • the first check valve 121 and the second check valve 122 in the centrifugal reaction microtube are designed It has a slope shape, and the slope of the second one-way valve 122 is greater than that of the first one-way valve 121.
  • the reaction reagent, the cleaning solution, and/or the specimen are isolated by the first one-way valve 121 and remain in the first reaction zone 111.
  • the reaction reagent, cleaning solution and/or sample will pass from the first reaction zone 111 through the first one-way valve 121 to the second reaction zone 112, and due to the second one-way valve 122 The slope is greater than the first one-way valve 121, so the reaction reagent, cleaning solution, and/or sample will not pass through the second one-way valve 122.
  • the reaction reagent, cleaning solution and/or specimen will pass from the second reaction zone 112 through the second one-way valve 122 with a relatively large slope without backflow To the first reaction zone 111.
  • the centrifugal reaction microtube 5 includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, and a second reaction zone.
  • the one-way valve 122 further includes a third reaction zone 113 and a third one-way valve 123.
  • the third reaction zone 113 can contain the reaction mixture for the third reaction, and the third one-way valve 123 is arranged between the third reaction zone 113 and the first reaction zone 111 to move the reaction mixture to the first reaction by means of the third centrifugal force. ⁇ 111.
  • a nucleic acid extraction reaction can be performed in the third reaction zone 113
  • a polymerase chain reaction can be performed in the first reaction zone 111
  • a nucleic acid hybridization reaction can be performed in the second reaction zone 112.
  • the second one-way valve 122 and the third one-way valve 123 are selected in descending order of the constant.
  • the third one-way valve 123 will open, while the first one-way valve 121 and the second one-way valve 122 are closed; at the speed suitable for the first one-way valve 121 to open Next, the first one-way valve 121 and the third one-way valve 123 will be opened, and the second one-way valve 122 will remain closed.
  • the centrifugal reaction microtube 6 includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, a second check valve 122, and a third reaction zone 111.
  • the waste liquid zone 13 is further included, which can be used to collect the waste liquid after the reaction.
  • the centrifugal reaction microtubes are preferably made of heat-resistant Made of optical materials.
  • Heat resistance means that the centrifugal reaction microtube does not change its properties, such as optical properties, at a certain temperature.
  • the specific temperature may be at least 120 degrees.
  • Optical material means that the material has the characteristics of low dispersion, low chromatic aberration, wide wavelength range of light penetration, low refractive index, and high transmittance. More preferably, the optical material may include quartz, glass or plastic.
  • a centrifugal reaction microtube is provided. As shown in FIG. 8, it includes a first reaction zone 111, a first one-way valve 121, a second reaction zone 112, a second one-way valve 122 and a waste liquid zone 13. After adding the reaction reagent or sample to the reaction microtube, the mouth of the tube can be capped to prevent the reagent or sample from splashing.
  • the length of the centrifugal reaction microtube can be 60 mm, and those skilled in the art can freely adjust the length according to actual needs and the centrifugal device used.
  • the length of the centrifugal reaction microtube can be 40mm; or when the centrifugal reaction microtube is implemented as in the fifth embodiment As shown in the example, with three reaction zones and three one-way valves, the length of the centrifugal reaction microtube can be 100mm.
  • the outside of the first reaction zone 111 can be adjacent to the heating module 14, which can raise the temperature of the first reaction zone 111 by means of substantial contact heating.
  • the heating module 14 may be U-shaped to effectively surround the first reaction zone 111, thereby increasing the heating speed of the first reaction zone 111.
  • a heating module can also be provided outside the first reaction zone 111 and the second reaction zone 112 at the same time to independently control the reaction temperature of the first reaction zone 111 and the second reaction zone 112.
  • the present application provides a centrifugal reaction device, including: a reaction centrifuge disk, which is inclined upwardly from an axis, and the reaction centrifuge disk has a plurality of symmetrically arranged fixing racks for accommodating a plurality of centrifuges.
  • Type reaction microtubules the reaction centrifuge disc presents a disc-like structure (that is, the center is low, the edges are high), and it is inclined upward and outward from the rotation axis, so that the reaction microtubes will naturally tilt after being placed in the reaction centrifuge disc, which helps the reaction microtubes The liquid flows naturally toward the axis of the reaction centrifugal disc due to the action of gravity.
  • the inclination of the reaction centrifuge disk from the axis of rotation to the edge may be about 1 to 89 degrees, preferably 10 to 80 degrees, 20 to 70 degrees, 30 to 60 degrees, 40 to 50 degrees, or any degree in between.
  • the reaction centrifugal plate can also be designed with a horizontal angle, and the above-mentioned purpose can be achieved by only matching with reaction microtubes with inclined inner surfaces.
  • the reaction microtubes in order to achieve high reaction throughput, can be designed to be arranged on the reaction centrifuge disk in an eight-row manner, as shown in FIG. 9.
  • the use of 8 reaction microtubes as a group is beneficial to increase the speed of adding reaction reagents or samples.
  • the reaction microtubes are arranged in the reaction centrifuge disk in 6 groups and 8 rows.
  • the reaction centrifuge disk can also adjust the reaction centrifuge disk according to actual needs and the reaction centrifuge disk used. The number of groups of 8 rows, such as 8 groups, 6 groups, 4 groups or 2 groups.
  • the centrifugal reaction device may further include a liquid separation module, which adds reaction reagents and/or samples into the centrifugal reaction microtubes.
  • the centrifugal reaction microtube is placed with the nozzle (the left side of the centrifugal reaction microtube 1 in Fig. 1) facing the direction of the rotation axis of the reaction centrifuge disc, so as to facilitate the separation module when adding reaction reagents and/or samples. There is no need to move in a large area, resulting in longer time required for liquid dispensing.
  • the centrifugal reaction device may further include a temperature control module for controlling the reaction temperature of the centrifugal reaction microtubes, including heating block, liquid (e.g., hot water), gas (e.g., hot air), Far infrared rays, etc.
  • a temperature control module for controlling the reaction temperature of the centrifugal reaction microtubes, including heating block, liquid (e.g., hot water), gas (e.g., hot air), Far infrared rays, etc.
  • the heating block and the liquid heating surround the centrifugal reaction microtubes in a U-shaped or O-shaped manner to increase the efficiency of temperature control.
  • the temperature of the reaction zone when the polymerase chain reaction is performed in the reaction zone, the temperature of the reaction zone must be controlled to be 0-100°C, preferably 4-95°C.
  • control the temperature of the reaction zone at a high temperature of 96°C to open the double-stranded DNA; during the conjugation phase, control the temperature of the reaction zone at about 60°C to allow the primers to pair with the template DNA; and
  • the temperature in the reaction zone was controlled at 72°C to extend the DNA, and the opening cycle was repeated several times.
  • the centrifugal reaction device may further include a signal detection module for detecting the reaction signal of the centrifugal reaction microtube.
  • the signal detection module can be a fluorescent, luminescent or visible light camera sensing system. After the reaction in the reaction zone of the centrifugal reaction microtube is completed, biomolecules (such as antibodies, aptamers, peptides or nucleic acids) labeled with fluorescence, luminescence or color signals can be added to the reaction zone to detect the Signal is qualitative or quantitative reaction product.
  • the centrifugal reaction device may further include a waste liquid collection module for collecting waste liquid leaving from the centrifugal reaction microtube.
  • a waste liquid collection module can be provided in the centrifugal reaction device to collect the waste liquid leaving the centrifugal reaction microtube.
  • the waste liquid collection module is a vacuum suction device arranged on the inner wall of the centrifugal reaction device. When the waste liquid leaves the centrifugal reaction microtubes 1, 3, and 5 from the one-way valve, the vacuum suction device will directly The waste liquid is sucked out without splashing back into the open one-way valve, causing reaction pollution.
  • the present application provides a centrifugal detection method, including: providing a centrifugal reaction microtube; and adding a sample and/or reaction reagent to the reaction zone to form a reaction mixture, and centrifuging the centrifugal reaction microtube To react.
  • the sample and/or reaction reagent are sequentially added to the centrifugal reaction microtube 3 to form a reaction mixture.
  • the centrifugal reaction microtube 3 is centrifuged to perform the first reaction in the first reaction zone 111.
  • the centrifugal speed or centrifugal force can be controlled, so that the first reaction is performed by shaking the reaction mixture when the first one-way valve 121 and the second one-way valve 122 are closed.
  • the centrifugal reaction microtube 3 is centrifuged at a speed with the first centrifugal force, and the reaction mixture passes through the opened first check valve 121 to the second reaction zone 112 for the second reaction.
  • the centrifugal reaction microtube 3 is centrifuged at a speed with a second centrifugal force, so that the reaction mixture leaves the second reaction zone 112 through the opened second one-way valve 122.
  • the cleaning solution can be added to the centrifugal reaction microtube 3 through the liquid separation module, and the centrifugal reaction microtube 3 can be centrifuged at a speed lower than the first centrifugal force and the second centrifugal force to wash by shaking.
  • the first reaction zone 111 or the second reaction zone 112 is performed.
  • the nucleic acid sample and the reaction reagent are added to the centrifugal reaction microtube with the liquid separation module.
  • sample tank such as a 1.5mL centrifuge tube or a 96-well plate
  • the PCR reaction is carried out in the temperature range of 37-60-95°C for about 40-45 cycles.
  • the concentration of nucleic acid was detected by the spectrophotometer in the centrifugal reaction device.
  • V Take 100 ⁇ L of hybridization reaction buffer (salt buffer) from the reagent tank (such as 1.5mL or 15mL centrifuge tube) and add it to the centrifuge reaction microtube;
  • the nucleic acid hybridization reaction is performed in the second reaction zone by centrifugal mixing and temperature control, and the washing step is performed.
  • High-speed centrifugation (such as 1500-4000g centrifugal force) to open the second one-way valve to remove all the reaction mixture from the second reaction zone to the waste liquid zone or the waste liquid collection module of the centrifugal reaction device;
  • V Take 100 ⁇ L of cleaning solution 2 (salt buffer) from the reagent tank (such as 1.5mL or 15mL centrifuge tube) and add it to the centrifugal reaction microtube, and centrifuge at a medium speed (such as 500-1000g centrifugal force) to make only the first
  • the one-way valve is opened, and the cleaning solution 2 is moved to the second reaction zone, and then the biochip is cleaned by centrifugation at an intermittent low speed (such as a centrifugal force of 10-100 g) for 3 minutes;
  • cleaning solution 3 salt buffer
  • reagent tank such as 1.5mL or 15mL centrifuge tube
  • medium speed such as 500-1000g centrifugal force
  • the one-way valve is opened, and the cleaning solution 3 is moved to the second reaction zone, and then the biochip is cleaned by centrifugation at an intermittent low speed (such as a centrifugal force of 10-100 g) for 3 minutes;
  • V Take 50 ⁇ L of the coloring agent (salt buffer) from the reagent tank (such as 1.5mL or 15mL centrifuge tube) and add it to the centrifugal reaction microtube, and centrifuge at a medium speed (such as 500-1000g centrifugal force) to make only the first Open the one-way valve to allow the color former to move to the second reaction zone, and then centrifuge at an intermittent low speed (such as a centrifugal force of 10-100 g) to react the color former with the biochip for 15 minutes;
  • a medium speed such as 500-1000g centrifugal force
  • an intermittent low speed such as a centrifugal force of 10-100 g
  • IX Take 100 ⁇ L of fixed terminator (salt buffer) from the reagent tank (such as 1.5mL or 15mL centrifuge tube) and add it to the centrifugal reaction microtube, and centrifuge at a medium speed (such as 500-1000g centrifugal force) to make it only Open the one-way valve to allow the fixed terminator to move to the second reaction zone, and then centrifuge at an intermittent low speed (such as 10-100 g centrifugal force) to react the fixed terminator with the biochip for 3 minutes;
  • a medium speed such as 500-1000g centrifugal force
  • XI Take 100 ⁇ L of cleaning solution 5 (salt buffer) from the reagent tank (such as 1.5mL or 15mL centrifuge tube) and add it to the centrifugal reaction microtube, and centrifuge at a medium speed (such as 500-1000g centrifugal force) to make the first
  • the one-way valve is opened, the cleaning solution 5 is moved to the second reaction zone, and then the biochip is cleaned by centrifugation at an intermittent low speed (such as a centrifugal force of 10-100g) for 3 minutes;
  • XIII Use the camera to take pictures and record sequentially according to the sample numbers on the biochip, and then analyze the results by the software and output a report. For example, if a biochip corresponding to a patient's specimen shows a color reaction in a specific test at a specific location, it means that a patient has a positive reaction to the chip analysis result of the specific test.
  • the centrifugal reaction device and the centrifugal inspection method described in the present application human intervention can be avoided, and the goal of one-click molecular biological detection can be achieved.
  • the molecular biology detection machine With the help of the control of centrifugal force and the application of centrifugal reaction microtubes, the molecular biology detection machine can be miniaturized, which saves energy and space, and can greatly shorten the detection time. In addition, there is no need to transfer between different machines during the reaction process, thereby achieving many goals such as simple and fast operation, safe and energy-saving, accurate and economical, and avoiding pollution.

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Abstract

一种离心式反应微管、离心式反应装置及其离心式检验方法，离心式反应微管（1）中单向阀（12）借助于离心力打开，利用离心力在同一离心式反应微管（1）中进行萃取、反应、清洗和/或侦测信号。

Description

离心式反应微管、离心式反应装置及其离心式检验方法 技术领域
本申请涉及一种实验用的反应微管和反应装置，特别是涉及一种通过一键式完成分子生物检测的反应微管和反应装置。
背景技术
随着生物技术领域的发展，通过生化和分子生物方法进行检测的需求日渐增加。虽然目前市面上已具有可同时进行核酸放大和分析的聚合酶连锁反应(PCR)仪，然而仍须搭配许多仪器，例如：电动移液机、核酸杂交反应器、清洗仪、信号判读仪等，才可完成所有的检测步骤。因此，检测过程常繁复耗时，并且在移液过程中还常有污染的风险。
当期望分析的核酸或检体极度复杂时，目前市售产品尚无法一键式即可完成所有分子生物检测，无法同时兼顾定性与定量分析。
发明内容
有鉴于上述先前技术的问题，本发明的目的就是在提供一种离心式反应微管和反应装置，通过反应微管的设计，利用离心即可于同一反应微管中进行萃取、反应、清洗和/或侦测信号，达到减少人力需求和污染的功效。
根据本发明的一个目的，本发明的一个实施例提供一种离心式反应微管，包括：
第一反应区，供容纳反应混合物进行第一反应；以及
第一单向阀，位于第一反应区的离心方向，其中第一单向阀借助于第一离心力打开而使反应混合物离开第一反应区。
优选地，所述离心式反应微管进一步包括：
第二反应区，通过第一单向阀与第一反应区分离，借助于 第一离心力使反应混合物移动至第二反应区进行第二反应；以及
第二单向阀，设置于第二反应区中第一单向阀的相反侧，其中第二单向阀借助于第二离心力打开使反应混合物离开第二反应区。
优选地，第二离心力大于第一离心力。
优选地，第一离心力和第二离心力可分别为1～80,000g，并且第二离心力大于第一离心力。
优选地，可通过电控或磁控控制第一单向阀和第二单向阀开闭。
优选地，离心式反应微管可进一步包含设置于第二反应区的生物芯片，其上涂布包括抗体、适体、胜肽或核酸的生物分子。
优选地，离心式反应微管可进一步包括废液区，借助于第二单向阀与第二反应区分离。
优选地，第一反应区可进行聚合酶连锁反应和/或第二反应区可进行核酸杂交反应。
优选地，所述离心式反应微管进一步包括：
第三反应区，供容纳反应混合物进行第三反应；以及
第三单向阀，设置于第三反应区与第一反应区之间，借助于第三离心力使反应混合物移动至第一反应区。
优选地，离心式反应微管实质上可由光学材料所制成。进一步佳地，光学材料可包括石英、玻璃或塑料。
根据本发明的另一目的，本发明的一个实施例提供一种离心式反应装置，包括：
反应离心盘，从轴心以斜度向上倾斜，反应离心盘上具有对称设置的多个固定架，供容置多个离心式反应微管。
优选地，斜度可为约1至89度。
优选地，离心式反应装置可进一步包括信号侦测模块，用于侦测离心式反应微管的反应信号。
优选地，离心式反应装置可进一步包括分液模块，分别于离心 式反应微管中加入反应试剂和/或检体。
优选地，离心式反应装置可进一步包括温度控制模块，用于控制离心式反应微管的反应温度。
优选地，离心式反应装置可进一步包括废液收集模块，用于收集从离心式反应微管离开的废液。
根据本发明的再一目的，本发明的一个实施例提供一种离心检测方法，包括：
提供离心式反应微管；以及
加入检体和/或反应试剂至第一反应区以形成反应混合物，离心离心式反应微管以进行第一反应。
优选地，所述方法可进一步包括加入清洗液并间歇离心离心式反应微管。
优选地，所述方法可进一步包括侦测离心式反应微管的反应信号。
优选地，所述方法可进一步包括控制离心式反应微管的反应温度。
优选地，所述方法可进一步包括收集从离心式反应微管离开的废液。
优选地，所述方法可进一步包括：
离心离心式反应微管，借助于第一离心力使反应混合物通过第一单向阀移动至第二反应区进行第二反应。
优选地，所述方法可进一步包括：
离心离心式反应微管，借助于第二离心力使反应混合物通过第二单向阀离开第二反应区。
优选地，第二离心力大于第一离心力。
优选地，第一离心力和第二离心力可分别为1～80,000g，并且第二离心力大于第一离心力。
优选地，可进一步通过电控或磁控控制第一单向阀和第二单向阀开闭。
优选地，第一反应区可进行聚合酶连锁反应和/或第二反应区可进行核酸杂交反应。
本发明的上述以及其它目的、特征与优点，在参照以下的详细说明与优选实施例和随附的附图后，将变得明显。
附图说明
在以下的详细描述中，为了解释本发明，提供了许多具体细节，以便能彻底理解所公开的实施方式。然而，显而易见的是，一个或多个的实施方式可以在没有所述具体细节的情况下实现。在其它情况中，为了简化附图，公知的结构和流程将以示意性的方式显示。
图1是根据本申请的第1实施例的离心式反应微管的示意图。
图2是根据本申请的第2实施例的离心式反应微管的示意图。
图3是根据本申请的第3实施例的离心式反应微管的示意图。
图4(A)-图4(C)是根据本申请的第3A实施例的离心式反应微管的使用状态示意图。
图5(A)、图5(B)是分别根据本申请的第4、4A实施例的离心式反应微管的示意图。
图6是根据本申请的第5实施例的离心式反应微管的示意图。
图7是根据本申请的第6实施例的离心式反应微管的示意图。
图8是根据本申请的一个优选实施例的离心式反应微管的示意图。
图9是根据本申请的一个实施例的反应离心盘的示意图。
具体实施方式
以下参照相关附图以详细描述实施例。然而，这些实施例可用不同方式来实现，但这并非实施或运用本发明的具体实施例的唯一形式，因此不应理解成对上述实施例的限制。实施方式中涵盖了多个具体实施例的特征以及用于建构与操作这些具体实施例的方法步骤与其顺序。然而，还可利用其他具体实施例来达成相同或均等的 功能与步骤顺序。相反的，提供这些实施例以使得本说明书可彻底且完整地进行公开，以充分地向本领域技术人员完全表达本发明的精神。附图中相似的组件符号是指相似的组件。在以下的叙述中，将不会详细描述公知的功能或结构，以不赘述实施例中不必要的细节。
除非另有定义，本文所用的所有技术用词与术语均与本领域技术人员所通常理解的意义相同。在发生冲突的情况下，以包括定义在内的本说明书为准。
在不和上下文冲突的情形下，本说明书所用的单数名词涵盖该名词的复数型；而所用的复数名词时还涵盖该名词的单数型。此外，在本说明书与权利要求中，“至少一”与“一或更多”等表述方式的意义相同，两者都代表包含了一、二、三或更多。
连接词“主要由……...组成”(consisting essentially of)用于界定组成物、方法或装置，其包括除明确指明以外的物料、步骤、特征、组分或组件，其限制条件是这些额外的物料、步骤、特征、组分或组件不会显著影响所要求保护的主题内容的基本与新颖特征。用语“主要由……组成”(consisting essentially of)居于“包含”(comprising)与“由……组成”(consisting of)之间的中间地带。
虽然用于界定本发明较广范围的数值范围与参数均为近似的数值，此处已尽可能精确地呈现具体实施例中的相关数值。然而，任何数值本质上不可避免地含有因个别测试方法所致的标准偏差。在此处，“约”通常是指实际数值在特定数值或范围的正负10％、5％、1％或0.5％之内。或者是，“约”一词代表实际数值落在平均值的可接受标准误差之内，根据本领域技术人员的考虑而定。除了实施例之外，或除非另有明确的说明，当可理解本文中所用的所有范围、数量、数值与百分比(例如用于描述材料用量、时间长短、温度、操作条件、数量比例和其他相似者)均经过“约”的修饰。因此，除非另有相反的说明，本说明书与权利要求所公开的数值参数均为近似的数值，并且可根据需求而改变。至少应将这些数值参数理解 为所指出的有效位数与套用一般进位法所得到的数值。在此处，将数值范围表示成由端点至另一端点或介于二端点之间；除非另有说明，此处所述的数值范围都包含端点。
在一个实施例中，提供一种离心式反应微管，其包括：至少反应区，供容纳包括反应试剂、清洗液和/或检体的反应混合物进行反应；以及至少单向阀，分别位于反应区的离心方向，借助于离心力使反应混合物朝向远离轴心方向流动，并在离心速度或离心力超过阈值时打开单向阀而使反应混合物离开反应区，并在离心速度或离心力低于阈值时使单向阀关闭，避免反应混合物回流至原反应区。也就是说，通过设置可选择性地反复开启或关闭的单向阀，可将反应微管区隔成至少一个反应区，供分析物(如蛋白质、核酸等生物分子)纯化、放大和分析等使用。
本申请中，“单向阀”是指可在未离心时控制反应试剂、清洗液和/或检体留在反应区中，并在离心时可使反应试剂、清洗液和/或检体单向通过所述“单向阀”至另一反应区而不回流的机构。
单向阀体的作动原理可以有多种方式，例如：机械式单向阀，其由不同弹性常数的弹簧与不同重量的圆珠所组成，材质可为金属或非金属。不同大小的离心力可对应不同弹性常数的阀体，让反应微管中的不同阀体在不同大小的离心力下开启或关闭。即配合离心力高低的运用，单向阀可控制反应混合物的滞留或排除。除此之外，单向阀也可由电控或磁控等方式，控制单向阀的开闭。
举例而言，若单向阀是由压缩弹簧控制其开闭，则在静止、没有离心或者离心力未达弹簧的压缩应力的状况下，压缩弹簧为完全延伸，而使单向阀完全密闭。当离心力到达弹簧的压缩应力时，随着离心力逐步增加，压缩弹簧则会因压缩而逐步变短，进而使单向阀打开，使反应混合物可以离开旋转轴心的方向移动通过单向阀。
所有反应过程中所产生的废液，可采用密闭收集或真空收集，根据反应微管有无开口以及废液量而定。在一个实施例中，废液采用密闭收集，即离心式反应微管可进一步包括废液区，借助于单向 阀与反应区分离，使在反应区进行反应后的反应混合物或者清洗液，借助于离心可离开反应区至废液区而不回流。
在一个实施例中，离心式反应微管的反应区中可设置生物芯片，其上涂布生物分子，供与反应混合物进行杂交。生物分子可包括抗体、适体、胜肽或核酸等。
如图1所示，在第1实施例中，离心式反应微管1包括一个反应区11和一个单向阀12。举例而言，经PCR放大后的核酸分子，可在反应区11进行与预先涂布目标核酸的生物芯片反应后，部分互补的核酸分子便会附着至生物芯片上，再通过离心的方式使经未附着的核酸分子通过单向阀12移除。
在第2实施例中，如图2所示，离心式反应微管2除包括一个反应区11和一个单向阀12外，进一步包括废液区13，可用于收集反应后的废液。
在第3实施例中，如图3所示，离心式反应微管3包括第一反应区111、第一单向阀121、第二反应区112和第二单向阀122。第一反应区111可供反应混合物进行第一反应；第一单向阀121位于第一反应区111的离心方向；第二反应区112通过第一单向阀121与第一反应区111分离；第二单向阀122设置于第二反应区112中第一单向阀121的相反侧。
在第3实施例中，由于具有两个反应区，因此能够在同一离心式反应微管3中依序进行两个反应。例如，在第一反应区111可进行聚合酶连锁反应和/或第二反应区112可进行核酸杂交反应；或在第一反应区111可进行核酸萃取和/或第二反应区112可进行核酸杂交反应。
在该实施例中，本领域技术人员能够根据需求选择不同或相同弹性常数的弹簧，以控制第一单向阀121和第二单向阀122开闭的弹簧在相同离心条件下开闭或者在不同离心条件下开闭。在一个优选实施例中，当使用相同弹性常数的弹簧时，可搭配不同重量的圆珠，即于第一单向阀121使用重量较重的圆珠，而在第二单向阀122 使用重量较轻的圆珠，因此开启第一单向阀121所需的离心力会小于开启第二单向阀122所需的离心力。因此在较低转速的情况下，仅有第一单向阀121会打开，第二单向阀122则是关闭的情况。在另一优选实施例中，当使用不同弹性常数的弹簧时，为避免第一反应区111的反应混合物离心时同时通过第一单向阀121和第二单向阀122而未停留于第二反应区112，将第二单向阀122的弹簧的弹性常数大于第一单向阀121的弹簧的弹性常数。在此情况下，相较于第一单向阀121，需要较大的离心力才能打开第二单向阀122，即需要较高的转速才能打开第二单向阀122。因此，在较低转速的情况下，仅有第一单向阀121会打开，第二单向阀122则是关闭的情况。
在一个优选实施例中，第一离心力和第二离心力可分别为1～80,000g，并且第二离心力大于第一离心力。
在第3A实施例中，如图4所示，离心式反应微管包括第一反应区111、第一单向阀121、第二反应区112和第二单向阀122。与第3实施例不同处为第一单向阀121和第二单向阀122由单一弹簧控制。如图4(A)所示，第一单向阀121、第二反应区112和第二单向阀122共同形成滑动件，可在离心时随离心力方向滑动。其中，滑动件可与弹簧相连，使滑动件在离心后，可回到静止状态的位置。在一个优选实施例中，弹簧可为压缩弹簧，设置于图中滑动件右侧；或者弹簧可为拉伸弹簧，设置于滑动件左侧。
举例而言，图4(A)为未进行的离心式反应微管，第一反应区111和第二反应区112并未连通，并且滑动件处于静止状态的第一位置。当以第一离心力进行离心时，滑动件便移动至(B)所示的第二位置，使通孔与第一单向阀121连通，反应试剂、清洗液和/或检体便会从第一反应区111通过第一单向阀121至第二反应区112，而第二单向阀122尚未与通孔连通，因此反应试剂、清洗液和/或检体不会通过第二单向阀122。当进一步以大于第一离心力的第二离心力进行离心时，滑动件便进一步移动至(C)所示的第三位置，使通孔与第二单向阀122连通，并且通孔不与第一单向阀121连通，因此反 应试剂、清洗液和/或检体便会从第二反应区112通过第二单向阀122排除，而不会回流至第一反应区111。
在第4实施例中，如图5(A)所示，离心式反应微管4除包括第一反应区111、第一单向阀121、第二反应区112和第二单向阀122外，进一步包括废液区13，可用于收集反应后的废液。
在第4A实施例中，如图5(B)所示，第4A实施例与第4实施例相比，离心式反应微管中的第一单向阀121和第二单向阀122是设计为斜坡状，并且第二单向阀122的斜度大于第一单向阀121。在该实施例中，未离心时，反应试剂、清洗液和/或检体通过第一单向阀121隔绝而留在第一反应区111中。当以第一离心力进行离心时，反应试剂、清洗液和/或检体便会从第一反应区111通过第一单向阀121至第二反应区112，而由于第二单向阀122的斜度大于第一单向阀121，因此反应试剂、清洗液和/或检体不会通过第二单向阀122。当进一步以大于第一离心力的第二离心力进行离心时，反应试剂、清洗液和/或检体便会从第二反应区112通过斜度较大的第二单向阀122，而不会回流至第一反应区111。
如图6所示，在第5实施例中，与第3实施例类似，只是离心式反应微管5除包括第一反应区111、第一单向阀121、第二反应区112和第二单向阀122外，进一步包括第三反应区113和第三单向阀123。第三反应区113可容纳反应混合物进行第三反应，而第三单向阀123设置于第三反应区113与第一反应区111之间，借助于第三离心力使反应混合物移动至第一反应区111。
在第5实施例中，由于具有三个反应区，因此能够在同一离心式反应微管5中依序进行三个反应。例如，在第三反应区113可进行核酸萃取反应，在第一反应区111可进行聚合酶连锁反应和/或第二反应区112可进行核酸杂交反应。
在一个优选实施例中，如前所述，为避免第三反应区113的反应混合物离心时一次通过第一单向阀121、第二单向阀122和第三单向阀123而未停留于第一反应区111，或者避免第一反应区111的反 应混合物离心时同时通过第一单向阀121和第二单向阀122而未停留于第二反应区112，将这些单向阀的弹性常数由大到小依序选择第二单向阀122、第一单向阀121和第三单向阀123。因此，在最低转速的情况下，仅有第三单向阀123会打开，第一单向阀121和第二单向阀122则是关闭的情况；在适合第一单向阀121打开的转速下，第一单向阀121和第三单向阀123会打开，第二单向阀122则是维持关闭的情况。
在第6实施例中，如图7所示，离心式反应微管6除包括第一反应区111、第一单向阀121、第二反应区112、第二单向阀122、第三反应区113和第三单向阀123外，进一步包括废液区13，可用于收集反应后的废液。
值得注意的是，由于须在离心式反应微管中进行多种反应，例如聚合酶连锁反应和核酸杂交反应，并且定性或定量侦测反应产物，因此离心式反应微管优选地可由耐热的光学材料所制成。“耐热”是指该离心式反应微管在某特定温度下不会改变其性质，例如光学性质。优选地，所述特定温度可至少为120度。“光学材料”是指在该材料具有色散低、色差低、光穿透的波长范围大、折射率小、穿透率高等特性。更优选地，所述光学材料可包括石英、玻璃或塑料。
在一个优选实施例中，提供一种离心式反应微管。如图8所示，其包括第一反应区111、第一单向阀121、第二反应区112、第二单向阀122和废液区13。在反应微管中加入反应试剂或检体后，可将管口封盖，以避免反应试剂或检体等液体溅出。所述离心式反应微管的长度可为60mm，对于本领域技术人员，可根据实际需求和所搭配使用的离心装置，而自由调整其长度。例如，当离心式反应微管如第1实施例所示，仅具有一个反应区和一个单向阀时，离心式反应微管的长度可为40mm；或者当离心式反应微管如第5实施例所示，具有三个反应区和三个单向阀时，离心式反应微管的长度可为100mm。
第一反应区111外侧可邻近加热模块14，其可通过实质接触加热的方式升高第一反应区111的温度。优选地，加热模块14可为U字型，以有效环绕第一反应区111，进而提高对第一反应区111的加热速度。相似地，还可同时设置加热模块于第一反应区111和第二反应区112外侧，以独立地控制第一反应区111和第二反应区112的反应温度。
在一个实施例中，本申请提供一种离心式反应装置，包括：反应离心盘，从轴心以斜度向上倾斜，反应离心盘上具有对称设置的多个固定架，供容置多个离心式反应微管。换句话说，反应离心盘呈现盘状结构(即中心低，边缘高)，从旋转轴心向上向外倾斜，使反应微管放入反应离心盘后会自然倾斜，有助于反应微管内的液体，因重力的作用朝向反应离心盘轴心方向自然流动。即当反应离心盘倾斜角度愈大，则反应混合物甩出后回流速度越快，可加速反应的进行，还可达到节能的目的。因离心作用而甩出的液体，在终止离心或离心力变小时，会借助于重力作用往低处流动，即朝反应离心盘旋转轴心方向回流，借助于间歇的离心，即可在无需附加任何移液设备情况下，达成液体快速交换或混合的目的。优选地，反应离心盘从旋转轴心至边缘斜度可为约1至89度，优选为10至80度、20至70度、30至60度、40至50度或其间的任意度数。可选地，所述反应离心盘还可设计为水平角度，仅需搭配内面倾斜的反应微管还可达成上述目的。
在一个优选实施例中，为达成反应的高通量，反应微管可设计为以八连排的方式设置于反应离心盘上，如图9所示。也就是说，以8个反应微管为一组的方式，有利于增加添加反应试剂或检体的速度。对于本领域技术人员，可根据实际需求和所搭配使用的离心装置，而自由调整一组反应微管中的个数，例如12连排、8连排、6连排、4连排或2连排等，不限于此。此外，在图9中，反应微管以6组8连排的方式排列于反应离心盘，本领域技术人员还可根据实际需求和所搭配使用的反应离心盘，而调整反应离心盘上可设置的8 连排的组数，例如8组、6组、4组或2组等。
在一个实施例中，离心式反应装置可进一步包括分液模块，分别于离心式反应微管中加入反应试剂和/或检体。离心式反应微管以管口(如图1中离心式反应微管1左侧)朝向反应离心盘旋转轴心的方向放置，以有利于分液模块在加入反应试剂和/或检体时，无须大范围移动，造成分液所需时间较长。
在一个实施例中，离心式反应装置可进一步包括温度控制模块，用于控制离心式反应微管的反应温度，包括通过加热块、液体(如，热水)、气体(如，热空气)、远红外线等。优选地，加热块和液体加热以U字型或O字型方式围绕离心式反应微管，以增加温度控制的效率。
例如，在反应区中进行聚合酶链锁反应时，须控制反应区的温度为0～100℃，优选为4～95℃。举例而言，在变性阶段时，将反应区温度控制于96℃高温下，使双股DNA打开；在接合阶段时，将反应区温度控制于约60℃下，让引子与模版DNA配对；以及在延伸阶段时，将反应区温度控制于在72℃，使DNA延伸，并且重复上开循环数次。
在一个实施例中，离心式反应装置可进一步包括信号侦测模块，用于侦测离心式反应微管的反应信号。例如，信号侦测模块可为荧光、冷光或可见光照相感测系统。在离心式反应微管的反应区中的反应完成后，可于反应区中添加标记荧光、冷光或颜色信号的生物分子(如抗体、适体、胜肽或核酸等)，以通过侦测该信号而定性或定量反应产物。
优选地，离心式反应装置可进一步包括废液收集模块，用于收集从离心式反应微管离开的废液。例如，在离心式反应微管的第1实施例、第3实施例和第5实施例中，离心式反应微管1、3、5都不具有废液区13，即无法在离心式反应微管1、3、5收集反应废液。在此情况下，可通过在离心式反应装置中设置废液收集模块而收集从离心式反应微管离开的废液。在一个优选实施例中，废液收集模 块为设置于离心式反应装置内壁上的真空吸引装置，当废液从单向阀离开离心式反应微管1、3、5时，真空吸引装置会直接将废液吸除，而不会回溅至开启的单向阀内，造成反应污染。
在又一实施例中，本申请提供一种离心检测方法，包括：提供离心式反应微管；以及加入检体和/或反应试剂至反应区以形成反应混合物，离心所述离心式反应微管以进行反应。
以下将参照图3说明根据本发明的一个实施例的离心检测方法。首先，在离心式反应微管3中依序加入检体和/或反应试剂，以形成反应混合物。离心该离心式反应微管3，以在第一反应区111进行第一反应。其中，可控制离心速度或离心力，使在第一单向阀121和第二单向阀122关闭的情况下震荡反应混合物进行该第一反应。接着，以具有第一离心力的速度离心离心式反应微管3，使反应混合物通过开启的第一单向阀121而到第二反应区112进行第二反应。以具有第二离心力的速度离心离心式反应微管3，使反应混合物通过开启的第二单向阀122而离开第二反应区112。可选地，可在反应过程中，通过分液模块将清洗液加入至离心式反应微管3，并以低于第一离心力和第二离心力的速度离心离心式反应微管3，以震荡清洗进行第一反应区111或第二反应区112。
离心检测方法的具体实施例：
(1)以分液模块将核酸检体和反应试剂加入至离心式反应微管。
I.从检体槽(如1.5mL离心管或96孔盘)取1-20μL的待测检体加入至离心式反应微管中；
II.从试剂槽(如1.5mL离心管)取1-20μL的试剂加入离心式反应微管；
III.从另一试剂槽(如1.5mL或15mL离心管)取20-30μL的矿物油加入离心式反应微管；以及
IV.低速(如10-100g的离心力)离心，将检体和反应试剂混合于第一反应区，第一单向阀和第二单向阀仍关闭。
(2)借助于离心混合与温度控制于第一反应区进行PCR，产生诸多扩增片段(Amplicon)。
以37-60-95℃的温度范围内进行PCR反应约40-45次循环反应。
(3)第一次信号侦测，进行核酸的定量分析。
以离心式反应装置中的分光亮度计侦测核酸浓度。
(4)以第一离心力进行离心，使扩增片段进入经过第一单向阀进入包含核酸生物芯片的第二反应区。
I.预热第二反应区至50-60℃；
II.从试剂槽(如1.5mL或15mL离心管)取20μL的变性液(强碱)，加入离心式反应微管；
III.低速(如10-100g的离心力)离心，让变性液穿过矿物油，并与原反应混合物混合，第一单向阀和第二单向阀仍关闭；
IV.静置30分钟，使DNA变性，由双股变单股，方便杂交反应进行；
V.从试剂槽(如1.5mL或15mL离心管)取100μL的杂交反应缓冲液(含盐缓冲液)加入离心式反应微管；
VI.低速(如10-100g的离心力)离心，使杂交反应缓冲液在第一反应区中与反应混合物混合，方便杂交反应进行，第一单向阀和第二单向阀仍关闭；以及
VII.中速(如500-1000g的离心力)离心，使仅第一单向阀开启，让前述混合物从第一反应区移至第二反应区。
(5)借助于离心混合与温度控制于第二反应区进行核酸杂交反应，并进行清洗步骤。
I.在一定温度(例如50℃)和时间(例如30-60min)下间歇低速(如10-100g的离心力)离心，以在离心力与重力的作用下 使得反应混合物可以在第二反应区中来回往复，以有利于杂交反应的进行；
II.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以将所有反应混合物从第二反应区移除至废液区或离心式反应装置的废液收集模块；
III.从试剂槽(如1.5mL或15mL离心管)取100μL的清洗液1(含盐缓冲液)加入离心式反应微管，先中速(如500-1000g的离心力)离心，使仅第一单向阀开启，让清洗液1移至第二反应区，接着再以间歇低速(如10-100g的离心力)离心清洗生物芯片3min；
IV.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以从第二反应区移除清洗液1；
V.从试剂槽(如1.5mL或15mL离心管)取100μL的清洗液2(含盐缓冲液)加入离心式反应微管，先中速(如500-1000g的离心力)离心，使仅第一单向阀开启，让清洗液2移至第二反应区，接着再以间歇低速(如10-100g的离心力)离心清洗生物芯片3min；
VI.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以从第二反应区移除清洗液2。
(6)第二次信号判读，进行核酸的定性分析。
I.从试剂槽(如1.5mL或15mL离心管)取50μL的含抗体的共轭液(含盐缓冲液)加入离心式反应微管，先中速(如500-1000g的离心力)离心，使仅第一单向阀开启，让共轭液移至第二反应区，接着再以间歇低速(如10-100g的离心力)离心使共轭液与生物芯片反应20min；
II.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以从第二反应区移除共轭液；
III.从试剂槽(如1.5mL或15mL离心管)取100μL的清洗液3(含 盐缓冲液)加入离心式反应微管，先中速(如500-1000g的离心力)离心，使仅第一单向阀开启，让清洗液3移至第二反应区，接着再以间歇低速(如10-100g的离心力)离心清洗生物芯片3min；
IV.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以从第二反应区移除清洗液3；
V.从试剂槽(如1.5mL或15mL离心管)取50μL的呈色剂(含盐缓冲液)加入离心式反应微管，先中速(如500-1000g的离心力)离心，使仅第一单向阀开启，让呈色剂移至第二反应区，接着再以间歇低速(如10-100g的离心力)离心使呈色剂与生物芯片反应15min；
VI.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以从第二反应区移除呈色剂；
VII.从试剂槽(如1.5mL或15mL离心管)取100μL的清洗液4(含盐缓冲液)加入离心式反应微管，先中速(如500-1000g的离心力)离心，使仅第一单向阀开启，让清洗液4移至第二反应区，接着再以间歇低速(如10-100g的离心力)离心清洗生物芯片3min；
VIII.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以从第二反应区移除清洗液4；
IX.从试剂槽(如1.5mL或15mL离心管)取100μL的固定终止剂(含盐缓冲液)加入离心式反应微管，先中速(如500-1000g的离心力)离心，使仅第一单向阀开启，让固定终止剂移至第二反应区，接着再以间歇低速(如10-100g的离心力)离心使固定终止剂与生物芯片反应3min；
X.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以从第二反应区移除固定终止剂；
XI.从试剂槽(如1.5mL或15mL离心管)取100μL的清洗液5(含盐缓冲液)加入离心式反应微管，先中速(如500-1000g的离 心力)离心，使仅第一单向阀开启，让清洗液5移至第二反应区，接着再以间歇低速(如10-100g的离心力)离心清洗生物芯片3min；
XII.高速(如1500-4000g的离心力)离心，使第二单向阀开启，以从第二反应区移除清洗液5；
XIII.利用相机根据生物芯片上的样本编号依序照相记录，再由软件分析结果，并输出报告。例如：某病人检体对应的生物芯片，在特定位置的特定试验出现呈色反应，即代表某病人对该特定试验的芯片分析结果呈阳性反应。
(7)完成检验。
如上所述，通过本申请所述的离心式反应微管、离心式反应装置及其离心式检验方法，可免除人力介入，而达成一键式完成分子生物检测的目的。借助于离心力的控制搭配离心式反应微管的应用，能够将分子生物检测机台微小化，既节能又省空间，可大幅缩短检测时长。此外，反应过程中无须在不同机台间的转移，进而达成了操作简便快速、安全节能、准确又经济实惠、避免污染等诸多目的。
以上所述仅为示例性，而非为限制性。任何未脱离本发明的精神与范畴，而对其进行的等效修改或变更，均应包含于权利要求所界定的范围中。

Claims (30)

  1. 一种离心式反应微管，包括：
    第一反应区，供容纳反应混合物进行第一反应；以及
    第一单向阀，位于所述第一反应区的离心方向，其中所述第一单向阀借助于第一离心力打开而使所述反应混合物离开所述第一反应区。
  2. 根据权利要求1所述的离心式反应微管，进一步包括：
    第二反应区，通过所述第一单向阀与所述第一反应区分离，借助于所述第一离心力使所述反应混合物移动至所述第二反应区进行第二反应；以及
    第二单向阀，设置于所述第二反应区中所述第一单向阀的相反侧，其中所述第二单向阀借助于第二离心力打开使所述反应混合物离开所述第二反应区。
  3. 根据权利要求2所述的离心式反应微管，其中所述第二离心力大于所述第一离心力。
  4. 根据权利要求2所述的离心式反应微管，其中所述第一离心力和所述第二离心力分别为1～80,000g，并且所述第二离心力大于所述第一离心力。
  5. 根据权利要求2所述的离心式反应微管，其中进一步通过电控或磁控控制所述第一单向阀和所述第二单向阀开闭。
  6. 根据权利要求2所述的离心式反应微管，进一步包含设置于所述第二反应区的生物芯片，所述生物芯片上涂布包括抗体、适体、胜肽或核酸的生物分子。
  7. 根据权利要求2所述的离心式反应微管，进一步包括废液区，借助于所述第二单向阀与所述第二反应区分离。
  8. 根据权利要求2所述的离心式反应微管，其中所述第一反应区进行聚合酶连锁反应。
  9. 根据权利要求8所述的离心式反应微管，其中所述第二反应 区进行核酸杂交反应。
  10. 根据权利要求2所述的离心式反应微管，进一步包括：
    第三反应区，供容纳所述反应混合物进行第三反应；以及
    第三单向阀，设置于所述第三反应区与所述第一反应区之间，借助于第三离心力使所述反应混合物移动至所述第一反应区。
  11. 根据权利要求1所述的离心式反应微管，其中所述离心式反应微管实质上由光学材料所制成。
  12. 根据权利要求11所述的离心式反应微管，其中所述光学材料包括石英、玻璃或塑料。
  13. 一种离心式反应装置，包括：
    反应离心盘，从轴心以一定斜度向上倾斜，所述反应离心盘上具有对称设置的多个固定架，供容置多个根据权利要求1至13中任一项所述的离心式反应微管。
  14. 根据权利要求13所述的离心式反应装置，其中所述斜度为约1至89度。
  15. 根据权利要求13所述的离心式反应装置，进一步包括信号侦测模块，侦测所述离心式反应微管的反应信号。
  16. 根据权利要求13所述的离心式反应装置，进一步包括分液模块，分别于所述离心式反应微管加入反应试剂和/或检体。
  17. 根据权利要求13所述的离心式反应装置，进一步包括温度控制模块，控制所述离心式反应微管的反应温度。
  18. 根据权利要求13所述的离心式反应装置，进一步包括废液收集模块，收集从所述离心式反应微管离开的废液。
  19. 一种离心检测方法，包括：
    提供根据权利要求1所述的离心式反应微管；以及
    加入检体和/或反应试剂至所述第一反应区以形成反应混合物，离心所述离心式反应微管以进行第一反应。
  20. 根据权利要求19所述的方法，进一步包括加入清洗液并间歇离心所述离心式反应微管。
  21. 根据权利要求19所述的方法，进一步包括侦测所述离心式反应微管的反应信号。
  22. 根据权利要求19所述的方法，进一步包括控制所述离心式反应微管的反应温度。
  23. 根据权利要求19所述的方法，进一步包括收集从所述离心式反应微管离开的废液。
  24. 根据权利要求19所述的方法，进一步包括：
    离心所述离心式反应微管，借助于第一离心力使所述反应混合物通过所述第一单向阀移动至第二反应区进行第二反应。
  25. 根据权利要求24所述的方法，进一步包括：
    离心所述离心式反应微管，借助于一第二离心力使所述反应混合物通过第二单向阀离开所述第二反应区。
  26. 根据权利要求25所述的方法，其中所述第二离心力大于所述第一离心力。
  27. 根据权利要求25所述的方法，其中所述第一离心力和所述第二离心力分别为1～80,000g，并且所述第二离心力大于所述第一离心力。
  28. 根据权利要求25所述的方法，其中进一步通过电控或磁控控制所述第一单向阀和所述第二单向阀开闭。
  29. 根据权利要求24所述的方法，其中所述第一反应区进行聚合酶连锁反应。
  30. 根据权利要求29所述的方法，其中所述第二反应区进行核酸杂交反应。
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