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WO2024092855A1 - 一种集成全封闭式核酸检测系统以及检测装置 - Google Patents

一种集成全封闭式核酸检测系统以及检测装置 Download PDF

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Publication number
WO2024092855A1
WO2024092855A1 PCT/CN2022/130409 CN2022130409W WO2024092855A1 WO 2024092855 A1 WO2024092855 A1 WO 2024092855A1 CN 2022130409 W CN2022130409 W CN 2022130409W WO 2024092855 A1 WO2024092855 A1 WO 2024092855A1
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Prior art keywords
nucleic acid
sample
detection
module
cavity
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PCT/CN2022/130409
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English (en)
French (fr)
Inventor
喻学锋
舒伟良
周文华
惠允
王占龙
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深圳先进技术研究院
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Publication of WO2024092855A1 publication Critical patent/WO2024092855A1/zh

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis

Definitions

  • the present invention relates to the fields of biomedical engineering and nucleic acid detection technology, and in particular to an integrated fully enclosed nucleic acid detection system and a detection device.
  • PCR Polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • RCA rolling circle amplification
  • RPA recombinant polymerase amplification
  • centrifugal column method or magnetic bead method is usually used for nucleic acid extraction, which generally requires lysis, binding, elution and other steps, making the entire "sample in result out" fully automated instrument very difficult to achieve.
  • manual transfer is mostly used in the prior art, which is not only cumbersome, time-consuming and labor-intensive, but also difficult to fully and efficiently transfer samples. Manual operation can easily lead to unstable results and difficulty in detection.
  • the mainstream technology for molecular detection is fluorescent quantitative PCR technology. Since PCR technology has the characteristics of exponential amplification templates, the existing open consumables make the entire operation process prone to PCR aerosol contamination.
  • Chinese patent CN200710158570.3 provides a liquid reservoir micropump fully integrated on a microfluidic chip, but the chip can only realize part of the functions of nucleic acid detection, and does not consider the actual needs of clinical users, so it is difficult to put it into practical use.
  • the purpose of the present invention is to provide an integrated fully enclosed nucleic acid detection system and detection device, aiming to solve the problem that the existing nucleic acid detection technology is basically based on the detection of fluorescence modules, the required optical path modules are complex and expensive, and the existing open consumables make the entire operation process prone to PCR aerosol contamination.
  • An integrated fully enclosed nucleic acid detection system comprising:
  • sample pre-processing module used for extracting nucleic acid from the sample to be tested, wherein the sample pre-processing module comprises at least one sample lysis unit, at least one nucleic acid binding unit, and at least one nucleic acid elution unit;
  • a nucleic acid amplification module connected to the sample pre-treatment module is used to amplify the nucleic acid in the sample to be tested obtained by extraction, and the nucleic acid amplification module includes at least one system preparation unit and at least one nucleic acid amplification unit;
  • An electrochemical detection module connected to the nucleic acid amplification module is used to detect the amplified nucleic acid, and the electrochemical detection module includes a detection electrode and a DNA probe for the sample to be tested;
  • a sample post-processing module connected to the sample pre-processing module, the nucleic acid amplification module and the electrochemical detection module, respectively, and used to receive waste liquid generated by each module;
  • a plurality of transfer units are respectively arranged between the sample pre-processing module and the nucleic acid amplification module, between the nucleic acid amplification module and the electrochemical detection module, between the sample post-processing module and the sample pre-processing module, between the nucleic acid amplification module and the electrochemical detection module, between the sample lysis unit, the nucleic acid binding unit and the nucleic acid elution unit in the sample pre-processing module, and between the system preparation unit and the nucleic acid amplification unit in the nucleic acid amplification module.
  • the integrated fully enclosed nucleic acid detection system wherein the nucleic acid detection system also includes a rotary valve, which controls the connection of the transfer unit of the nucleic acid detection system.
  • the integrated fully enclosed nucleic acid detection system further comprises a computer control module for connecting and controlling the sample pre-processing module, the nucleic acid amplification module, the electrochemical detection module and the sample post-processing module respectively.
  • nucleic acid detection device comprises a housing and three structural layers installed in the housing, the structural layers comprising a main body layer, a flow channel layer, and an electrode layer, and the different structural layers are sealed by gluing or ultrasonic bonding;
  • the main body layer includes a plurality of chambers for sample pre-treatment, in which reagents for sample pre-treatment are pre-installed; a freeze-dried reagent chamber for nucleic acid amplification, in which freeze-dried powder reagent balls for nucleic acid amplification are pre-installed; and a syringe chamber for placing a syringe, which is used to aspirate reagents;
  • the flow channel layer includes a plurality of channels for sample pre-processing and a plurality of channels for sample post-processing;
  • the electrode layer includes a PCR reaction area for nucleic acid amplification, a detection electrode for electrochemical detection, and a DNA probe for a sample to be tested;
  • the nucleic acid detection device also includes a rotary valve.
  • the integrated fully enclosed nucleic acid detection device wherein the main body layer is formed by plastic injection molding; the several chambers used for sample pretreatment include a sample port, a mixing pool, a lysis liquid chamber, a cleaning liquid I chamber, a cleaning liquid II chamber, and an elution liquid chamber; the pre-packaged reagents are individually packaged and placed in the corresponding chamber positions of the main body layer, and the freeze-dried powder reagent balls are placed in the freeze-dried reagent chambers.
  • the flow channel layer is formed by plastic injection molding;
  • the several passages for sample pre-treatment include a sample port passage, a mixing pool passage, a lysis liquid passage, a cleaning liquid I passage, a cleaning liquid II passage and an eluent passage;
  • the several passages for sample post-treatment include several waste liquid passages;
  • the flow channel layer also includes a syringe passage; the upper surface of the flow channel layer is connected to the chamber of the main layer, the flow channel structure is formed on the lower surface, and the lower surface is sealed with a single-sided tape.
  • the integrated fully enclosed nucleic acid detection device wherein the PCR reaction area for nucleic acid amplification includes a PCR reaction chamber, a PCR mixed solution inlet and a PCR mixed solution outlet.
  • the integrated fully enclosed nucleic acid detection device wherein the detection electrode includes a counter electrode, a reference electrode and a working electrode.
  • the integrated fully enclosed nucleic acid detection device wherein the DNA probe is preset on the surface of the working electrode, one end of the DNA probe is connected to the surface of the working electrode, and the other end is connected to ferrocene; the DNA probe self-assembles on the surface of the working electrode through Au-S bonds to form a uniformly distributed target DNA capture area.
  • the sample port and the syringe cavity are connected by rotating the rotary valve to suck the sample to be tested into the syringe;
  • the rotary valve is used to connect the injector cavity and the lysing liquid cavity, so that the lysing liquid is sucked into the injector and mixed with the sample to be tested, thereby lysing the sample to be tested;
  • the binding solution is sucked into the injector by the rotation control of the rotary valve, and the nucleic acid in the lysed sample to be tested is adsorbed by the nucleic acid capture magnetic beads in the binding solution;
  • the injector cavity and the cleaning solution I cavity are connected by the rotation control of the rotary valve, and the cleaning solution I is sucked into the injector to clean the nucleic acid capture magnetic beads;
  • the injector cavity and the cleaning liquid II cavity are connected by rotating the rotary valve, and the cleaning liquid II is sucked into the injector to clean the nucleic acid capture magnetic beads again;
  • the injector cavity and the eluent cavity are connected by rotating the rotary valve, and the eluent is sucked into the injector to elute the nucleic acid in the nucleic acid capture magnetic beads;
  • the injector cavity and the freeze-dried reagent cavity are connected by rotating the rotary valve, the eluent containing the nucleic acid is mixed with the freeze-dried reagent to prepare a PCR reaction solution, and then the PCR reaction solution is completely sucked into the injector cavity;
  • the PCR reaction solution is distributed to the PCR reaction chamber through the rotation control of the rotary valve to perform a PCR reaction and amplify the nucleic acid;
  • the DNA probe captures the amplified nucleic acid and outputs the detection result through the detection electrode.
  • the present invention provides an integrated fully enclosed nucleic acid detection system and detection device, which integrates a sample pre-treatment module, a nucleic acid amplification module and an electrochemical detection module, and can realize full integration of complex samples from pre-treatment to detection, saving the pre-treatment time of the detection.
  • the present invention can realize fully enclosed nucleic acid extraction and PCR detection. After the sample is placed, sample lysis, nucleic acid binding, cleaning, elution and PCR detection are realized in a completely closed state, ensuring the airtightness of the nucleic acid extraction and expansion process, reducing the interference of external aerosols on the test results, and preventing the infection hazard of samples to the test personnel, realizing a fully automated detection process of sample input and result output.
  • the present invention adopts highly sensitive electrochemical detection, does not require a complex optical detection module, and can also realize the detection of the target object more sensitively.
  • the present invention greatly reduces the setting of the flow path switch control valve through the design of an integrated rotary valve, simplifies the structure of the chip, reduces the required area of the chip, and thus reduces the production cost.
  • FIG1 is a schematic diagram of a module of an integrated fully enclosed nucleic acid detection system provided in an embodiment of the present invention.
  • Figure 2 is a schematic diagram of the three-layer structural module layout of an integrated fully enclosed nucleic acid detection device provided by an embodiment of the present invention.
  • FIG3 is a schematic diagram of the main layer structure of an integrated fully enclosed nucleic acid detection device provided in an embodiment of the present invention.
  • FIG4 is a schematic diagram of the flow channel layer structure of an integrated fully enclosed nucleic acid detection device provided in an embodiment of the present invention.
  • FIG5 is a schematic diagram of the electrode layer structure of an integrated fully enclosed nucleic acid detection device provided in an embodiment of the present invention.
  • FIG6 is a schematic diagram of the structure of an integrated fully enclosed nucleic acid detection device provided in an embodiment of the present invention.
  • the present invention provides an integrated fully enclosed nucleic acid detection system and a detection device.
  • a detection device In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
  • the directions or positional relationships indicated by the terms “above”, “below”, “left”, “right”, etc. are based on the directions or positional relationships shown in the drawings, or are the directions or positional relationships in which the invented product is usually placed when in use. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as limiting the present invention.
  • the terms “first”, “second”, and “third” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.
  • the terms “installed”, “connected”, and “connected” should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal communication of two components.
  • installed should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal communication of two components.
  • An embodiment of the present invention provides an integrated fully enclosed nucleic acid detection system, comprising:
  • sample pre-processing module used for extracting nucleic acid from the sample to be tested, wherein the sample pre-processing module comprises at least one sample lysis unit, at least one nucleic acid binding unit, and at least one nucleic acid elution unit;
  • a nucleic acid amplification module connected to the sample pre-treatment module is used to amplify the nucleic acid in the sample to be tested obtained by extraction, and the nucleic acid amplification module includes at least one system preparation unit and at least one nucleic acid amplification unit;
  • An electrochemical detection module connected to the nucleic acid amplification module is used to detect the amplified nucleic acid, and the electrochemical detection module includes a detection electrode and a DNA probe for the sample to be tested;
  • the sample post-processing module is connected to the sample pre-processing module, the nucleic acid amplification module and the electrochemical detection module respectively, and is used to receive waste liquid generated by each module.
  • the module schematic diagram of the nucleic acid detection system of the present invention is shown in Figure 1.
  • the nucleic acid detection system also includes multiple transfer units, which are respectively arranged between the sample pre-processing module and the nucleic acid amplification module, between the nucleic acid amplification module and the nucleic acid detection module, between the sample post-processing module and the sample pre-processing module, the nucleic acid amplification module and the electrochemical detection module, between the sample lysis unit, the nucleic acid binding unit and the nucleic acid elution unit in the sample pre-processing module, and between the system preparation unit and the nucleic acid amplification unit in the nucleic acid amplification module.
  • the sample lysis unit lyses the sample to be tested, and includes a lysis solution and a device for placing the lysis solution, for example, a lysis solution chamber or a lysis solution pool; after the lysis solution is pre-packaged, it can be placed in the lysis solution chamber or the lysis solution pool, wherein one side of the pre-packaged sealing film is a puncturable sealing film, which is placed downward in the lysis solution chamber or the lysis solution pool.
  • the nucleic acid binding unit binds the nucleic acid in the sample to be tested, and includes a binding liquid and a device for placing the binding liquid, for example, it can be a binding liquid chamber or a mixing pool; the binding liquid contains magnetic beads that can capture nucleic acids; after the binding liquid is pre-packaged, it can be placed in the binding liquid chamber or the mixing pool, wherein one side of the pre-packaged sealing film is puncturable, and it is placed downward in the binding liquid chamber or the mixing pool.
  • a binding liquid and a device for placing the binding liquid for example, it can be a binding liquid chamber or a mixing pool; the binding liquid contains magnetic beads that can capture nucleic acids; after the binding liquid is pre-packaged, it can be placed in the binding liquid chamber or the mixing pool, wherein one side of the pre-packaged sealing film is puncturable, and it is placed downward in the binding liquid chamber or the mixing pool.
  • the nucleic acid elution unit elutes the nucleic acid bound to the magnetic beads, and includes an eluent and a device for placing the eluent, for example, an eluent chamber or an eluent pool; after the eluent is pre-packaged, it can be placed in the eluent chamber or the eluent pool, wherein one side of the pre-packaged sealing film is a puncturable sealing film, which is placed downward in the eluent chamber or the eluent pool.
  • the sample pre-treatment unit may also include a high temperature inactivation device, an automatic code scanning device, an automatic cover opening/closing device, a sealing cover, etc.
  • the sealing cover has breathable cotton to prevent virus diffusion and is connected to the atmosphere, which is conducive to balancing the cavity pressure after the liquid flows.
  • the function of the sample pre-treatment module is to crack the sample to be tested and extract the nucleic acid in the sample.
  • the collected samples include sputum, tissue, blood, feces, etc.
  • the pre-treatment of these samples is relatively complicated and requires compatibility with multiple treatment methods, such as chemical lysis solution guanidine isothiocyanate, biological enzyme lysis solution proteinase K, and heating and mechanical shearing, according to different sample configurations.
  • the system provided by the present invention can be compatible with various forms of samples, such as urine, blood, nasopharyngeal swabs, sputum, tissue, feces and other samples, and the reagent formula and increase or decrease the processing steps can be adjusted according to the specific sample.
  • the nucleic acid amplification module is used to amplify the nucleic acid extracted from the sample to be tested, and includes at least one system preparation unit and at least one nucleic acid amplification unit.
  • the system preparation unit is used to prepare a PCR reaction solution, including PCR reaction reagents and a reagent placement device.
  • a solution containing PCR reaction reagents can be pre-made into a lyophilized powder and placed in a lyophilized powder chamber; when a solution containing nucleic acid enters the lyophilized powder chamber, the lyophilized powder is dissolved, and the nucleic acid and PCR reaction reagent are mixed.
  • the lyophilized powder contains necessary reagents for PCR amplification reaction, such as enzymes, substrates, buffers, etc.
  • the nucleic acid amplification unit is used to perform a PCR reaction to amplify nucleic acid, and includes a PCR reaction chamber and components necessary for the PCR reaction, such as a temperature control component.
  • each unit forms an independent airtight space.
  • the existing technology is basically based on the detection of fluorescence modules, and the required optical path modules are relatively complex and expensive.
  • the present invention adopts highly sensitive electrochemical detection, which can start working by simply connecting to a USB port. It is very feasible for the parallel operation of multiple systems of a single instrument.
  • the nucleic acid detection system further comprises a rotary valve, which controls the connection of the transfer unit of the nucleic acid detection system.
  • the embodiment of the present invention greatly reduces the setting of the flow path switch control valve through the design of the integrated rotary valve, realizes the connection between the liquid chamber and the reaction chamber through the rotation of the rotary valve, simplifies the structure of the chip, reduces the required area of the chip, and thus reduces the production cost of the detection system.
  • the nucleic acid detection system further includes a computer control module for connecting and controlling the sample pre-processing module, the nucleic acid amplification module, the electrochemical detection module, and the sample post-processing module, respectively.
  • the nucleic acid detection system also includes a power assembly, which includes a first motor, a second motor and a micropump fixedly installed inside the shell, the first motor drives the rotary valve to rotate through a gear set, the second motor drives the syringe to suck liquid reagent through pressure, and the micropump is connected to the air inlet of the nucleic acid detection system through an air path.
  • a power assembly which includes a first motor, a second motor and a micropump fixedly installed inside the shell, the first motor drives the rotary valve to rotate through a gear set, the second motor drives the syringe to suck liquid reagent through pressure, and the micropump is connected to the air inlet of the nucleic acid detection system through an air path.
  • the present invention provides a fully enclosed high-sensitivity electrochemical detection system for nucleic acid extraction and amplification, which has extremely high integration and is equipped with the required nucleic acid extraction and detection reagents. After the sample is added, fully enclosed automatic processing is realized, realizing an automated device of "sample in, result out”. At the same time, it has excellent airtightness.
  • This is a fully integrated nucleic acid detection microfluidic chip that can accurately control the fluid and has very good airtightness. It is not connected to the outside air during use, which can greatly avoid the influence of the external environment on the test results and protect the health of the test personnel.
  • the electrochemical detection module includes a detection electrode and a DNA probe for the sample to be tested.
  • the present invention uses highly sensitive electrochemical detection, does not require a complex optical detection module, and can also detect the target more sensitively.
  • the electrode surface uses a special electroplating process to form a Christmas tree nanostructure, which greatly increases the specific surface area and improves the sensitivity of the electrode to the target substance.
  • the DNA probe modified with thiol and ferrocene self-assembles on the surface of the gold electrode through Au-S bonds to form a uniformly distributed target DNA capture area. Since the ferrocene on the DNA probe is close to the surface of the gold electrode, a relatively large peak current signal is generated.
  • the DNA probe hybridizes with the target DNA to form a DNA double strand with greater rigidity, so that the ferrocene at the tail end of the DNA probe is away from the gold electrode surface, which reduces the peak current in the square wave voltammetry method and constructs a "turn-off" type electrochemical sensor.
  • the electrochemical detection module of the detection system can also be changed to a fluorescence detection module, changing the electrode-based signal detection to a detection method based on fluorescence signals.
  • the embodiment of the present invention also provides an integrated fully enclosed nucleic acid detection device, which includes a shell and three structural layers installed in the shell, wherein the structural layers include a main body layer, a flow channel layer, and an electrode layer, and the different structural layers are sealed by gluing or ultrasonic bonding.
  • Figure 2 is a schematic diagram of the three-layer structural layer of the integrated fully enclosed nucleic acid detection device provided by the embodiment of the present invention.
  • the integrated fully enclosed nucleic acid detection device provided by the present invention is composed of a three-layer structure, namely a main body layer, a flow channel layer and an electrode layer.
  • the different structural layers are sealed by gluing or ultrasonic bonding.
  • All plastic parts of the detection device are produced by injection molding, and the materials used for injection molding include but are not limited to PMMA, PC, PS, COC, COP, etc.
  • the injection molding method can achieve the advantages of small batch difference, high output and low cost for mass production.
  • the electrode layer includes a detection electrode and a DNA probe for the sample to be tested.
  • the nucleic acid detection device further comprises a rotary valve, and the connection between the flow channel layers is controlled by the rotary valve.
  • the rotary valve uses a polyurethane or silicone rubber gasket to achieve sealing by compression.
  • the sealing of the rotary valve is the most critical part.
  • the sealing gasket of the rotary valve and the flow channel layer uses a polyurethane gasket or a silicone rubber gasket.
  • the present invention greatly reduces the setting of the flow path switch control valve through the design of an integrated rotary valve, and realizes the connection between the liquid chamber and the reaction chamber through the rotation of the rotary valve, which simplifies the structure of the chip, reduces the required area of the chip, and thus reduces the production cost.
  • the use of the rotary valve greatly reduces the fluid switch control unit, reduces the processing area of the device, and reduces the processing cost.
  • the main body layer is formed by plastic injection molding, and the materials used for injection molding include but are not limited to PMMA, PC, PS, COC, COP, etc.
  • the main body layer includes several chambers for sample pre-treatment, in which reagents for sample pre-treatment are pre-set; a freeze-dried reagent chamber for nucleic acid amplification, in which freeze-dried powder reagent balls for nucleic acid amplification are pre-set; and a syringe chamber for placing a syringe, and the syringe is used to aspirate reagents;
  • the several chambers for sample pretreatment include a sample port, a mixing tank, a lysis liquid chamber, a cleaning liquid I chamber, a cleaning liquid II chamber, and an elution liquid chamber; the pre-packaged reagents are individually packaged and placed in corresponding chamber positions of the main layer, and the freeze-dried powder reagent balls are placed in the freeze-dried reagent chamber.
  • FIG3 is a schematic diagram of the structure of the main layer of the nucleic acid detection device.
  • the main layer includes a sample port, a mixing tank, a lysate chamber, a cleaning liquid I chamber, a cleaning liquid II chamber, an elution chamber, and a syringe chamber.
  • the sample port is used to add the sample to be tested, and the mixing tank is used to perform a mixing reaction of the corresponding solution.
  • the pre-packaged reagents are individually packaged and placed in the corresponding chamber positions of the main layer, for example, the lysate is placed in the lysate chamber, the cleaning liquid I is placed in the cleaning liquid I chamber, the cleaning liquid II is placed in the cleaning liquid II chamber, and the elution chamber is placed.
  • the sealing film that can be punctured is placed downwardly in the corresponding chamber position of the main layer.
  • the syringe chamber is used to place the syringe, and the syringe is used to aspirate or mix liquids.
  • the main body layer further comprises a freeze-dried reagent chamber, and the freeze-dried powder reagent ball is directly placed in the freeze-dried reagent chamber.
  • the freeze-dried powder contains necessary reagents for PCR amplification reaction, such as enzymes, substrates, buffers, etc.
  • the main body layer further includes a waste liquid cavity, and the waste liquid cavity is internally filled with high-strength absorbent cotton.
  • the position, quantity and shape of the preset reagent chambers can be changed and adjusted according to specific needs.
  • the flow channel layer is formed by plastic injection molding, and the materials used for injection molding include but are not limited to PMMA, PC, PS, COC, COP, etc.
  • the flow channel layer is sealed with 3M single-sided medical PCR special pressure sensitive adhesive.
  • the flow channel layer includes several channels for sample pre-processing and several channels for sample post-processing.
  • the several passages for sample pre-processing include a sample port passage, a mixing pool passage, a lysis liquid passage, a cleaning liquid I passage, a cleaning liquid II passage and an eluent passage;
  • the several passages for sample post-processing include several waste liquid passages;
  • the flow channel layer also includes a syringe passage; the upper surface of the flow channel layer is connected to the chamber of the main layer, the flow channel structure is formed on the lower surface, and the lower surface is sealed with a single-sided tape.
  • FIG4 is a schematic diagram of the structure of the flow channel layer of the nucleic acid detection device.
  • the flow channel layer includes a sample port passage, a mixing pool passage, a lysate passage, a cleaning liquid I passage, a cleaning liquid II passage, an eluent passage, a waste liquid passage, and a syringe passage; and also includes a passage for a freeze-dried reagent.
  • the upper surface of the flow channel layer is connected to the chamber of the main body layer, and the flow channel structure is formed on the lower surface, which is sealed with a single-sided adhesive.
  • the electrode layer includes a PCR reaction area for nucleic acid amplification, a detection electrode for electrochemical detection, and a DNA probe for a sample to be tested.
  • the PCR reaction area for nucleic acid amplification includes a PCR reaction chamber, a PCR mixed solution inlet and a PCR mixed solution outlet.
  • FIG. 5 is a schematic diagram of the structure of the electrode layer of the nucleic acid detection device.
  • the electrode layer also includes a PCR reaction chamber, a PCR mixed solution inlet and a PCR mixed solution outlet.
  • the electrode layer is sealed with 3M single-sided medical PCR special pressure sensitive adhesive.
  • the detection electrode includes a counter electrode, a reference electrode, and a working electrode.
  • the DNA probe is preset on the surface of the working electrode, one end of the DNA probe is connected to the surface of the working electrode, and the other end is connected to ferrocene; the DNA probe self-assembles on the surface of the working electrode through Au-S bonds to form a uniformly distributed target DNA capture area.
  • the detection electrode is a printed electrode.
  • the number of detection electrodes can be changed according to the detection target, and can be reduced to a design of 1 to 8.
  • the present invention uses sputtering or printing to make a gold electrode on an injection-molded substrate, and then forms a three-dimensional Christmas tree nanostructure on the surface of the working electrode through a special electroplating process, which can greatly increase the specific surface area of the electrode.
  • the ferrocene DNA probe self-assembles on the surface of the gold electrode through Au-S bonds to form a uniformly distributed target DNA capture area.
  • the electrode layer and the flow channel layer are sealed with double-sided tape, the PCR reaction chamber is located on the upper surface of the electrode layer, and the liquid in the flow channel layer enters through the through hole of the electrode layer.
  • a single-sided pressure-sensitive adhesive is used for sealing, and no pressure is applied to the connection position between the PCR reaction chamber and the through hole of the electrode layer, so that it is in an unsealed state; when liquid enters the through hole, the liquid can enter the PCR reaction chamber along the unsealed area, and then flow out from the unsealed area at the other end.
  • the application of highly sensitive electrochemical detection in the embodiments of the present invention can improve the sensitivity of detection and reduce the manufacturing cost of the supporting detection instrument, and does not require high skills from the user.
  • the surface of the detection electrode adopts a unique electroplating process, which greatly increases the specific surface area of the working electrode and enhances the detection sensitivity of the electrode.
  • the present invention provides a fully enclosed high-sensitivity electrochemical detection device for nucleic acid extraction and amplification, which has extremely high integration and is equipped with the required nucleic acid extraction and detection reagents. After the sample is added, it realizes fully enclosed automatic processing, realizing an automated device of "sample in, result out”. At the same time, it has excellent airtightness.
  • This is a fully integrated nucleic acid detection microfluidic chip that can accurately control the fluid and has very good airtightness. It is not connected to the outside air during use, which can greatly avoid the influence of the external environment on the test results and protect the health of the test personnel.
  • the embodiment of the present invention further provides a detection method of an integrated fully enclosed nucleic acid detection device, comprising the steps of:
  • the binding solution is sucked into the injector by rotating the rotary valve, and the nucleic acid in the lysed sample to be tested is adsorbed by the nucleic acid capture magnetic beads in the binding solution;
  • the DNA probe captures the amplified nucleic acid and outputs the detection result through the detection electrode.
  • the method provided by the present invention is based on the above-mentioned integrated fully enclosed nucleic acid detection device. Because the detection device is equipped with the required nucleic acid extraction and detection reagents, after the sample is added, fully enclosed automatic processing is realized, realizing "sample in and result out"; at the same time, it has excellent airtightness. This is a fully integrated nucleic acid detection microfluidic chip that can accurately manipulate the fluid, and the airtightness is very good. It is not connected to the outside air during use, which can greatly avoid the influence of the external environment on the test results, and can also protect the health of the test personnel. The method of the present invention can realize fully enclosed nucleic acid extraction and PCR detection.
  • sample lysis, nucleic acid binding, cleaning, elution and PCR detection are realized in a completely closed state, ensuring the airtightness of the nucleic acid extraction and expansion process, reducing the interference of external aerosols on the test results, and preventing the infection hazard of samples to the test personnel, realizing a fully automated detection process of sample in and result out.
  • Figure 6 shows one of the integrated fully enclosed nucleic acid detection devices provided in this embodiment, including a sample port for adding a sample to be tested; a mixing tank for performing a mixing reaction of corresponding solutions; a syringe for extracting and mixing different liquids; a rotary valve for controlling the connectivity of each chamber; chambers corresponding to the lysis solution, cleaning solution I, cleaning solution II, eluent and freeze-dried reagent; a waste liquid chamber with built-in absorbent cotton; a PCR reaction chamber for performing a PCR reaction of the nucleic acid of the sample to be tested; and a detection electrode.
  • the present invention is suitable for a variety of clinical samples, such as urine, blood, nasopharyngeal swabs, sputum, tissues, feces and other samples, and the reagent formula can be adjusted and the processing steps can be increased or decreased according to the specific samples.
  • the liquid is completely pumped into the syringe chamber, and then the rotary valve is turned to the mixing pool, the mixing pool and the syringe chamber are connected, and an equal volume of binding liquid (containing magnetic beads that capture nucleic acids) is pumped in.
  • the liquid is mixed evenly by suction with a syringe, and the mixing time is 1-15 minutes.
  • a strong electromagnet is placed at the bottom of the syringe cavity, the magnetic beads are fixed at the bottom of the syringe cavity, and then the rotary valve is turned to the waste liquid cavity, the waste liquid cavity and the syringe cavity are connected, and the syringe is pushed to squeeze the liquid into the waste liquid cavity (built-in high-strength absorbent cotton).
  • the rotary valve is turned to the cleaning liquid I cavity, the cleaning liquid I cavity and the syringe cavity are connected, 100-2000 ⁇ l of cleaning liquid I is pumped in, and the syringe is used to pump back and forth to fully mix for 1-10min.
  • a strong electromagnet is placed at the bottom of the syringe cavity, the magnetic beads are fixed at the bottom of the syringe cavity, the rotary valve is turned to the waste liquid cavity, the waste liquid cavity and the syringe cavity are connected, and the syringe is pushed to squeeze the liquid into the waste liquid cavity.
  • the rotary valve is turned to the cleaning liquid II cavity, the cleaning liquid II cavity and the syringe cavity are connected, 100-2000 ⁇ l of cleaning liquid II is pumped in, and the syringe is used to pump back and forth to fully mix for 1-10min.
  • a strong electromagnet is placed at the bottom of the syringe cavity, and the magnetic beads are fixed at the bottom of the syringe. Then the rotary valve is turned to the freeze-dried powder cavity, the freeze-dried powder cavity and the syringe cavity are connected, and a fixed volume of eluent is pushed into the freeze-dried powder cavity. Then the rotary valve is turned to the eluent cavity, the eluent cavity and the syringe cavity are connected, and the remaining liquid is pushed into the eluent cavity for standby use.
  • the rotary valve is turned to the freeze-dried powder cavity, the freeze-dried powder cavity and the syringe cavity are connected, and the PCR reaction liquid is mixed evenly by suction back and forth with the syringe, and finally all of it is sucked into the syringe cavity.
  • the rotary valve is turned to the PCR flow channel hole to connect the PCR flow channel and the injector cavity, and the injector is slowly pushed, and the PCR mixed liquid enters the PCR cavity along the flow channel.
  • the PCR cavity and the flow channel are not directly connected by a channel.
  • a single-sided pressure-sensitive adhesive is used as a sealing layer. When pressure is applied to seal, no pressure is applied to the connection between the liquid inlet and outlet and the PCR cavity, and other non-flow path areas are completely sealed, so that the flow path area is in a non-sealed state.
  • liquid enters from the PCR mixed liquid inlet it can enter the PCR cavity from the unsealed flow path area, and then enter the waste liquid flow path outlet from the PCR cavity through the unsealed flow path area, and the excess liquid finally enters the waste liquid cavity.
  • the PCR amplification reaction is carried out under the control of the external instrument.
  • the amplification reaction can be PCR, LAMP, SDA or RPA.
  • the DNA probe modified with thiol and ferrocene self-assembles on the surface of the gold electrode through Au-S bonds to form a uniformly distributed target DNA capture area. Since the ferrocene on the DNA probe is close to the surface of the gold electrode, a relatively large peak current signal is generated.
  • the DNA probe hybridizes with the target DNA to form a DNA double strand with greater rigidity, so that the ferrocene at the tail end of the DNA probe is away from the gold electrode surface, reducing the peak current in the square wave voltammetry method, and constructing a "turn-off" type electrochemical sensor.
  • the present invention provides an integrated fully enclosed nucleic acid detection system and detection device, the system includes a sample pre-treatment module, a nucleic acid amplification module, an electrochemical detection module and a rotary valve.
  • the device of the present invention integrates a sample collection port, a filter membrane, a fluid control rotary valve, a closed injector, etc., which can realize full integration from pre-treatment to detection of complex samples, saving the pre-treatment time of detection.
  • the present invention can realize fully enclosed nucleic acid extraction and PCR detection.
  • the present invention adopts highly sensitive electrochemical detection, does not require a complex optical detection module, and can also realize the detection of the target object more sensitively.
  • the electrode surface adopts a special electroplating process to form a Christmas tree nanostructure, which greatly increases the specific surface area and improves the sensitivity of the electrode to the target substance.
  • the present invention greatly reduces the number of flow path switch control valves through the design of an integrated rotary valve, simplifies the chip structure, reduces the required chip area, and thus reduces production costs.

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Abstract

一种集成全封闭式核酸检测系统以及检测装置，所述系统包括样本前处理模块、核酸扩增模块、电化学检测模块以及旋转阀。可对复杂样本从前处理到检测实现全集成，节省前处理时间。可实现全封闭式核酸提取及PCR 检测，保证了核酸提取及扩张过程的密闭性，减少外界气溶胶对检测结果的干扰，以及防止样本对检测人员的传染危害，实现了样本进结果出的全自动化检测过程。同时，采用高灵敏的电化学检测，不需要复杂的光学检测模块，而且还可以更加灵敏的实现对目标物的检测。不仅如此，通过集成旋转阀的设计，极大的缩减流路开关控制阀门的设置，简化了芯片的结构，降低了芯片的需求面积，从而缩减了生产成本。

Description

一种集成全封闭式核酸检测系统以及检测装置 技术领域
本发明涉及生物医学工程以及核酸检测技术领域，尤其涉及一种集成全封闭式核酸检测系统以及检测装置。
背景技术
近年来，由高致病性病原微生物(如新型冠状病毒、埃博拉病毒等)引起的新发、突发传染病不断出现，严重威胁到人体健康与公共卫生安全。早期、快速和准确的疾病检测对于最大限度地提高感染性疾病的危机管理效率和治疗效果至关重要。然而，目前的检测主要局限于集中中央实验室，通常样本会被带到医院或诊所进行检测，检测结果会在几天内反馈，在发展中国家或地区，由于缺乏熟练的人员和医疗基础设施，疾病检测往往会更久。因此，对便携、易于使用和即时诊断检测(POCT)的需求正在迅速增加。
分子诊断学正在革新感染性疾病的临床检测，其中一个显著的进步是减少了诊断感染性疾病所需的时间。聚合酶链反应(PCR)是最常用的分子诊断技术，但热循环需要一个耗能的仪器，为了简化及缩短热循环的时间，有发展出各种等温扩增反应，如环介导等温扩增(LAMP)、滚环扩增(RCA)和重组聚合酶扩增(RPA)，在这些技术的基础上，开发了许多商业化试剂盒，可以实现高灵敏和快速的检查病原体。然而，这些试剂盒在样本制备和核酸提取方面的操作步骤较多，限制了其在临床诊断中的广泛应用。因此，开发一种将分子诊断的所有步骤都集成到一个小型化设备中的片上实验室系统已成为一种趋势。在POCT诊断中，患者可以自己收集样本，无需医务人员的帮助，采集的样本可以在采样点立即进行分析和疾病筛查，而且只需要非常小的样本量就可以实现对生物标志物的检测。
在现有技术中，通常使用离心柱法或磁珠法进行核酸提取，一般需要进行裂解、结合、洗脱等步骤，使得整个“样本进结果出”的全自动化仪器非常难以实现。就各步骤中有效成分的转移而言，现有技术中多采用手动转移的方式，不仅操作繁琐、费时费力，且样本很难充分、高效第转移，人工操作极易导致结果不稳定，检测实现难度大。此外，分子检测的主流技术为荧光定量PCR技术， 由于PCR技术具有指数级扩增模板的特点，现有的开放式耗材使整个操作过程容易造成PCR气溶胶污染。例如中国专利CN200710158570.3，提供了一种完全集成于微流控芯片上的储液池微泵，但该芯片只能实现核酸检测的部分功能，且没有考虑临床用户的实际需求，很难实用化。
因此，现有技术还有待改进。
发明内容
鉴于上述现有技术的不足，本发明的目的在于提供一种集成全封闭式核酸检测系统以及检测装置，旨在解决现有核酸检测技术基本上基于荧光模块的检测，所需配备的光路模块复杂昂贵，且现有的开放式耗材使整个操作过程容易造成PCR气溶胶污染的问题，通过集成全封闭式核酸提取和扩增的电化学检测装置，最终实现对复杂样本从前处理到检测全集成，实现样本进结果出的全自动化检测过程。
本发明的技术方案如下：
一种集成全封闭式核酸检测系统，其中，包括：
样本前处理模块，用于提取待测样本中的核酸，所述样本前处理模块包括至少一个样本裂解单元，至少一个核酸结合单元，至少一个核酸洗脱单元；
与所述样本前处理模块连接的核酸扩增模块，用于对提取得到的待测样本中的核酸进行扩增，所述核酸扩增模块包括至少一个体系配制单元，至少一个核酸扩增单元；
与所述核酸扩增模块连接的电化学检测模块，用于对扩增后的核酸进行检测，所述电化学检测模块包括检测电极以及针对待测样本的DNA探针；
样本后处理模块，与所述样本前处理模块、所述核酸扩增模块以及所述电化学检测模块分别连接，用于接收各个模块产生的废液；
以及多个传递单元，分别设置在所述样本前处理模块与所述核酸扩增模块之间，所述核酸扩增模块与所述电化学检测模块之间，所述样本后处理模块与所述样本前处理模块之间、所述核酸扩增模块以及所述电化学检测模块之间，所述样本前处理模块中样本裂解单元、核酸结合单元以及核酸洗脱单元之间，所述核酸扩增模块中体系配制单元与核酸扩增单元之间。
所述的集成全封闭式核酸检测系统，其中，所述核酸检测系统还包括旋转阀，所述旋转阀控制所述核酸检测系统的传递单元的连接。
所述的集成全封闭式核酸检测系统，其中，所述核酸检测系统还包括计算机控制模块，用于对样本前处理模块、核酸扩增模块、电化学检测模块以及样本后处理模块分别进行连接与控制。
一种集成全封闭式核酸检测装置，其中，所述核酸检测装置包括壳体以及安装在壳体内的三层结构层，所述结构层包括主体层、流道层、电极层，不同结构层之间使用胶粘或超声键合的方式实现封接；
所述主体层包括用于样本前处理的若干腔室，其中预置用于样本前处理的试剂；还包括用于核酸扩增的冻干试剂腔，其中预置用于核酸扩增的冻干粉试剂球；还包括注塞器腔，用于放置注塞器，所述注塞器用于抽吸试剂；
所述流道层包括用于样本前处理的若干通路，用于样本后处理的若干通路；
所述电极层包括用于核酸扩增的PCR反应区，用于电化学检测的检测电极以及针对待测样本的DNA探针；
所述核酸检测装置还包括旋转阀。
所述的集成全封闭式核酸检测装置，其中，所述主体层采用塑料注塑成型；所述用于样本前处理的若干腔室包括样本口、混合池、裂解液腔、清洗液I腔、清洗液II腔以及洗脱液腔；预封装后的试剂单独封装后放置在主体层的对应腔室位置，冻干粉试剂球放置在冻干试剂腔。
所述的集成全封闭式核酸检测装置，其中，所述流道层采用塑料注塑成型；所述用于样本前处理的若干通路包括样本口通路、混合池通路、裂解液通路、清洗液I通路、清洗液II通路以及洗脱液通路；所述用于样本后处理的若干通路包括若干废液通路；所述流道层还包括注塞器通路；所述流道层上表面与主体层的腔室连通，流道结构在下表面成型，下表面使用单面胶进行封闭。
所述的集成全封闭式核酸检测装置，其中，所述用于核酸扩增的PCR反应区包括PCR反应腔、PCR混合液入口以及PCR混合液出口。
所述的集成全封闭式核酸检测装置，其中，所述检测电极包括对电极、参考电极以及工作电极。
所述的集成全封闭式核酸检测装置，其中，所述DNA探针预设在所述工作电极表面，DNA探针的一端接在工作电极表面，另一端连接二茂铁；所述DNA探针通过Au-S键在工作电极表面自组装形成均匀分布的目标DNA捕获区。
一种如上任一所述的集成全封闭式核酸检测装置的检测方法，其中，包括步骤：
对待测样本进行液化，并将液化后的待测样本加入到样本口；
通过旋转阀的旋转控制连通样本口和注塞器腔，将待测样本吸入注塞器；
通过旋转阀的旋转控制连通注塞器腔和裂解液腔，将裂解液吸入注塞器中和待测样本混合，对待测样本进行裂解；
通过旋转阀的旋转控制将结合液吸入注塞器，通过结合液中的核酸捕获磁珠吸附裂解后的待测样本中的核酸；
通过旋转阀的旋转控制连通注塞器腔和清洗液I腔，将清洗液I吸入注塞器，对核酸捕获磁珠进行清洗；
通过旋转阀的旋转控制连通注塞器腔和清洗液II腔，将清洗液II吸入注塞器，对核酸捕获磁珠进行再次清洗；
通过旋转阀的旋转控制连通注塞器腔和洗脱液腔，将洗脱液吸入注塞器，对核酸捕获磁珠中的核酸进行洗脱；
通过旋转阀的旋转控制连通注塞器腔和冻干试剂腔，将包含核酸的洗脱液与冻干试剂混合，配制成PCR反应液，然后将PCR反应液全部吸入注塞器腔；
通过旋转阀的旋转控制将PCR反应液分配至PCR反应腔，进行PCR反应，对核酸进行扩增；
DNA探针对扩增后的核酸进行捕获，并通过检测电极输出检测结果。
有益效果：本发明提供了一种集成全封闭式核酸检测系统以及检测装置，集成了样本前处理模块、核酸扩增模块以及电化学检测模块，可以对复杂样本从前处理到检测实现全集成，节省检测的前处理时间。本发明可实现全封闭式核酸提取及PCR检测，从样本放置完成后，就在完全封闭的状态实现样本裂解、核酸结合、清洗、洗脱以及PCR检测，保证了核酸提取及扩张过程的密闭性，减少外界气溶胶对检测结果的干扰，以及防止样本对检测人员的传染危害，实现了样本进结果出的全自动化检测过程。同时，本发明采用高灵敏的电化学检测，不需要复杂的光学检测模块，而且还可以更加灵敏的实现对目标物的检测。不仅如此， 本发明通过集成旋转阀的设计，极大的缩减流路开关控制阀门的设置，简化了芯片的结构，降低了芯片的需求面积，从而缩减了生产成本。
附图说明
图1为本发明实施例提供的一种集成全封闭式核酸检测系统的模块示意图。
图2为本发明实施例提供的一种集成全封闭式核酸检测装置的三层结构模块布局示意图。
图3为本发明实施例提供的集成全封闭式核酸检测装置的主体层结构示意图。
图4为本发明实施例提供的集成全封闭式核酸检测装置的流道层结构示意图。
图5为本发明实施例提供的集成全封闭式核酸检测装置的电极层结构示意图。
图6为本发明实施例提供的一种集成全封闭式核酸检测装置的结构示意图。
具体实施方式
本发明提供一种集成全封闭式核酸检测系统以及检测装置，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
需要说明的是，本发明中，术语“上方”、“下方”、“左”、“右”等指示的方位或位置关系为基于附图所示的方位或位置关系，或者是该发明产品使用时惯常摆放的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。此外，术语“第一”、“第二”、“第三”仅用于描述目的，而不能理解为指示或暗示相对重要性。
需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本实用新型中的具体含义。
需要说明的是，相似的标号和字母在下面的附图中表示类似项，因此，一旦某一项在一个附图中被定义，则在随后的附图中不需要对其进行进一步定义和解释，另外，在本发明中，仅是对技术方案的主要部件进行了相应的标注说明，其他附图上所体现的没有标注的零部件也属于本发明技术方案的一部分，且本发明中附图不清晰及附图中未标记的部分对本发明的技术方案没有影响，且都为本领域技术人员所公知，因此不能理解为对本发明的限制。
本发明实施例提供一种集成全封闭式核酸检测系统，包括：
样本前处理模块，用于提取待测样本中的核酸，所述样本前处理模块包括至少一个样本裂解单元，至少一个核酸结合单元，至少一个核酸洗脱单元；
与所述样本前处理模块连接的核酸扩增模块，用于对提取得到的待测样本中的核酸进行扩增，所述核酸扩增模块包括至少一个体系配制单元，至少一个核酸扩增单元；
与所述核酸扩增模块连接的电化学检测模块，用于对扩增后的核酸进行检测，所述电化学检测模块包括检测电极以及针对待测样本的DNA探针；
样本后处理模块，与所述样本前处理模块、所述核酸扩增模块以及所述电化学检测模块分别连接，用于接收各个模块产生的废液。
本发明的核酸检测系统的模块示意图如图1所示，所述核酸检测系统还包括多个传递单元，分别设置在所述样本前处理模块与所述核酸扩增模块之间，所述核酸扩增模块与所述核酸检测模块之间，所述样本后处理模块与所述样本前处理模块、所述核酸扩增模块以及所述电化学检测模块之间，所述样本前处理模块中样本裂解单元、核酸结合单元以及核酸洗脱单元之间，所述核酸扩增模块中体系配制单元与核酸扩增单元之间。
在一些实施方式中，所述样本裂解单元对待测样本进行裂解，其包括裂解液，以及裂解液的放置装置，例如，可以为裂解液腔或者裂解液池；裂解液进行预封装之后，可置于所述裂解液腔或者裂解液池内，其中预封装时有一面为可被穿刺的封膜，将此向下放置在所述裂解液腔或者裂解液池内。
在一些实施方式中，所述核酸结合单元对待测样本中的核酸进行结合，其包括结合液，以及结合液的放置装置，例如，可以为结合液腔或者混合池；结合液里面包含可以捕获核酸的磁珠；结合液进行预封装之后，可置于所述结合液腔或 者混合池内，其中预封装时有一面为可被穿刺的封膜，将此向下放置在所述结合液腔或者混合池内。
在一些实施方式中，所述核酸洗脱单元对磁珠结合的核酸进行洗脱，其包括洗脱液，以及洗脱液的放置装置，例如，可以为洗脱液腔或者洗脱液池；洗脱液进行预封装之后，可置于所述洗脱液腔或者洗脱液池内，其中预封装时有一面为可被穿刺的封膜，将此向下放置在所述洗脱液腔或者洗脱液池内。
在一些实施方式中，所述样本前处理单元还可以包括高温灭活装置、自动扫码装置、自动开/关盖装置、密封盖等等。所述密封盖带有透气棉，可以防止病毒扩散，并接通大气，有利于平衡液体流动后的腔体压力。
样本前处理模块的功能是裂解待测样本并提取样本中的核酸。采集的样本包括痰液、组织、血液、粪便等，这些样本的前处理是比较复杂的，需要兼容多种处理方式，如化学裂解液异硫氰酸胍、生物酶裂解液蛋白酶K、以及加热和机械剪切，根据不同的样本配置不同的方式。本发明提供的系统可以兼容多种形式的样本，如尿液、血液、鼻咽拭子、痰液、组织、粪便等样本，可以根据具体的样本调整试剂配方及增减处理步骤。
在一些实施方式中，核酸扩增模块用于对提取得到的待测样本中的核酸进行扩增，包括至少一个体系配制单元，至少一个核酸扩增单元。
在一些实施方式中，所述体系配制单元用于PCR反应液的配制，包括PCR反应试剂以及试剂的放置装置。例如，可将包含PCR反应试剂的溶液预先制成冻干粉，并放置在冻干粉腔；待包含了核酸的溶液进入到冻干粉腔时，溶解冻干粉，则核酸与PCR反应试剂进行混合。具体的，所述冻干粉包含了进行PCR扩增反应的必须试剂，例如酶、底物、缓冲液等等。
在一些实施方式中，所述核酸扩增单元用于进行PCR反应对核酸进行扩增，其包括PCR反应腔以及PCR反应必备的元件，例如温控元件等。
在一些实施方式中，每个单元各自形成独立的气密性空间。
现有技术基本是基于荧光模块的检测，所需配备的光路模块比较复杂昂贵，本发明采用了高灵敏的电化学检测，使用时只需接入USB插口就可以开始工作，对于单仪器的多个系统并行操作，具有非常高的可行性。
在一些实施方式中，所述核酸检测系统还包括旋转阀，所述旋转阀控制所述核酸检测系统的传递单元的连接。本发明实施例通过集成旋转阀的设计，极大的 缩减流路开关控制阀门的设置，通过旋转阀的转动实现液体腔与反应腔的联通，简化了芯片的结构，降低了芯片的需求面积，从而缩减了检测系统的生产成本。
在一些实施方式中，所述核酸检测系统还包括计算机控制模块，用于对样本前处理模块、核酸扩增模块、电化学检测模块以及样本后处理模块分别进行连接与控制。
在一些实施方式中，所述核酸检测系统还包括动力组件，所述动力组件包括固定安装在壳体内部的第一电机、第二电机和微泵，所述第一电机通过齿轮组驱动旋转阀旋转，所述第二电机通过压力驱动注塞器抽吸液体试剂，所述微泵通过气路与核酸检测系统的进气口连通。
本发明提供的是一种全封闭式核酸提取及扩增的高灵敏度电化学检测系统，具有极高的集成性，配备了所需的核酸提取和检测试剂，样本添加后，实现全封闭自动话处理，实现了“样本进结果出”的自动化装置。同时兼具极好的密闭性，这是一种全集成的核酸检测微流控芯片，可对流体进行精确的操控，密闭性非常好，使用过程中不予外部空气连通，能极大地避免外部环境对检测结果的影响，也能保护检测人员的健康。
在一些实施方式中，所述电化学检测模块包括检测电极以及针对待测样本的DNA探针。本发明采用高灵敏的电化学检测，不需要复杂的光学检测模块，而且还可以更加灵敏的实现对目标物的检测。电极表面采用特殊的电镀工艺形成圣诞树纳米结构，极大地增加了比表面积，提高了电极对目标物质的灵敏度。
修饰有巯基和二茂铁的DNA探针通过Au-S键在金电极表面自组装形成均匀分布的目标DNA捕获区，由于DNA探针上的二茂铁与金电极表面距离较近从而产生一个相对较大的峰电流信号。当加入目标DNA后，DNA探针与目标DNA杂化形成刚度较大的DNA双链，从而使DNA探针尾端的二茂铁远离金电极表面，使得方波伏安法中的峰电流减小，构建“turn-off”型电化学传感器。
在一些实施方式中，检测系统的电化学检测模块还可以变更为荧光检测模块，把基于电极的信号检测变更为依据荧光信号的检测方法。
本发明实施例还提供一种集成全封闭式核酸检测装置，所述核酸检测装置包括壳体以及安装在壳体内的三层结构层，所述结构层包括主体层、流道层、电极层，不同结构层之间使用胶粘或超声键合的方式实现封接。图2所示即为本发明实施例提供的集成全封闭式核酸检测装置的三层结构层示意图。
本发明提供的集成全封闭式核酸检测装置共由3层结构组成，分别是主体层、流道层以及电极层，不同结构层之间使用胶粘或超声键合的方式实现封接。检测装置的所有塑料部件都采用注塑成型的方式生产，注塑使用的材料包括但不限于PMMA、PC、PS、COC、COP等等。注塑成型的方式对于批量化生产能做到批间差小、产量高、成本低的优势。在一些实施方式中，所述电极层包括检测电极以及针对待测样本的DNA探针。
在一些实施方式中，所述核酸检测装置还包括旋转阀，通过旋转阀控制流道层之间的连接。
在一些实施方式中，所述旋转阀采用聚氨酯或硅橡胶垫圈使用压紧的方式实现密封。旋转阀的密封是最关键的地方，本发明中旋转阀与流道层的密封垫使用聚氨酯垫圈或硅橡胶垫圈。本发明通过集成旋转阀的设计，极大的缩减流路开关控制阀门的设置，通过旋转阀的转动实现液体腔与反应腔的联通，简化了芯片的结构，降低了芯片的需求面积，从而缩减了生产成本。旋转阀的使用大大减少了流体开关控制单元，缩减了装置的加工面积，降低了加工的成本。
在一些实施方式中，所述主体层采用塑料注塑成型，注塑使用的材料包括但不限于PMMA、PC、PS、COC、COP等等。所述主体层包括用于样本前处理的若干腔室，其中预置用于样本前处理的试剂；还包括用于核酸扩增的冻干试剂腔，其中预置用于核酸扩增的冻干粉试剂球；还包括注塞器腔，用于放置注塞器，所述注塞器用于抽吸试剂；
在一些具体的实施方式中，所述用于样本前处理的若干腔室包括样本口、混合池、裂解液腔、清洗液I腔、清洗液II腔以及洗脱液腔；预封装后的试剂单独封装后放置在主体层的对应腔室位置，冻干粉试剂球放置在冻干试剂腔。
图3所示为所述核酸检测装置的主体层的结构示意图。所述主体层包括样本口、混合池、裂解液腔、清洗液I腔、清洗液II腔、洗脱液腔、以及注塞器腔。样本口用于待测样本的加入，混合池用于进行相应溶液的混合反应。预封装后的试剂单独封装后放置在主体层的对应腔室位置，例如裂解液放置于裂解液腔，清洗液I放置于清洗液I腔，清洗液II放置于清洗液II腔，洗脱液放置于洗脱液腔。具体的，预封装试剂单独封装后，其可被穿刺的封膜向下放置在主体层的对应腔室位置。注塞器腔用于放置注塞器，注塞器用于抽吸或者混合液体。
在一些实施方式中，所述主体层还包括冻干试剂腔，冻干粉试剂球直接放置在冻干试剂腔。具体的，所述冻干粉包含了进行PCR扩增反应的必须试剂，例如酶、底物、缓冲液等等。
在一些实施方式中，所述主体层还包括废液腔，所述废液腔内置高强力吸水棉。
在另一些实施方式中，预置试剂腔的位置、数量及形状可以根据具体的需求进行变更调整。
在一些实施方式中，所述流道层采用塑料注塑成型，注塑使用的材料包括但不限于PMMA、PC、PS、COC、COP等等。所述流道层3M单面医用PCR专用压力敏感胶封闭。所述流道层包括用于样本前处理的若干通路，用于样本后处理的若干通路。
在一些具体的实施方式中，所述用于样本前处理的若干通路包括样本口通路、混合池通路、裂解液通路、清洗液I通路、清洗液II通路以及洗脱液通路；所述用于样本后处理的若干通路包括若干废液通路；所述流道层还包括注塞器通路；所述流道层上表面与主体层的腔室连通，流道结构在下表面成型，下表面使用单面胶进行封闭。
图4所示为所述核酸检测装置的流道层的结构示意图。所述流道层包括样本口通路、混合池通路、裂解液通路、清洗液I通路、清洗液II通路、洗脱液通路、废液通路以及注塞器通路；还包括冻干试剂的通路。所述流道层上表面与主体层的腔室连通，流道结构在下表面成型，下表面使用单面胶进行封闭。
在一些实施方式中，所述电极层包括用于核酸扩增的PCR反应区，用于电化学检测的检测电极以及针对待测样本的DNA探针。
在一些具体的实施方式中，所述用于核酸扩增的PCR反应区包括PCR反应腔、PCR混合液入口以及PCR混合液出口。
图5所示即为所述核酸检测装置的电极层的结构示意图，除检测电极以及DNA探针之外，所述电极层还包括PCR反应腔、PCR混合液入口以及PCR混合液出口。所述电极层通过3M单面医用PCR专用压力敏感胶封闭。
在一些实施方式中，所述检测电极包括对电极、参考电极以及工作电极。所述DNA探针预设在所述工作电极表面，DNA探针的一端接在工作电极表面，另 一端连接二茂铁；所述DNA探针通过Au-S键在工作电极表面自组装形成均匀分布的目标DNA捕获区。
在一些实施方式中，所述检测电极为印刷电极。
在另一些实施方式中，检测电极的数量可以根据检测目标物进行变更，可以是减少为1到8个的设计。
本发明在注塑好的基板上使用溅射或印刷的方式制作金电极，再经过特殊的电镀工艺，在工作电极表面形成了一层三维的圣诞树纳米结构，这样可以大大提高电极的比表面积。二茂铁DNA探针通过Au-S键在金电极表面自组装形成均匀分布的目标DNA捕获区。电极层与流道层通过双面胶进行封接，PCR反应腔位于电极层的上表面，流道层的液体通过电极层的通孔进入。在电极层上表面形成封闭层的时候使用单面压力敏感胶进行封闭，在PCR反应腔与电极层通孔的连线位置不施加压力，使其处于未封接状态；当通孔有液体进入时，液体可以沿着未封接的区域进入PCR反应腔，然后从另一端的未封接区域流出。
本发明实施例中高灵敏的电化学检测的应用，可提高检测的灵敏度，并降低配套检测仪器的制造成本，对于使用者的技能要求不高。检测电极表面采用独特的电镀工艺，大大提高了工作电极的比表面积，增强了电极的检测灵敏度。
本发明提供的是一种全封闭式核酸提取及扩增的高灵敏度电化学检测装置，具有极高的集成性，配备了所需的核酸提取和检测试剂，样本添加后，实现全封闭自动话处理，实现了“样本进结果出”的自动化装置。同时兼具极好的密闭性，这是一种全集成的核酸检测微流控芯片，可对流体进行精确的操控，密闭性非常好，使用过程中不予外部空气连通，能极大地避免外部环境对检测结果的影响，也能保护检测人员的健康。
本发明实施例还提供一种集成全封闭式核酸检测装置的检测方法，包括步骤：
S10、对待测样本进行液化，并将液化后的待测样本加入到样本口；
S20、通过旋转阀的旋转控制连通样本口和注塞器腔，将待测样本吸入注塞器；
S30、通过旋转阀的旋转控制连通注塞器腔和裂解液腔，将裂解液吸入注塞器中和待测样本混合，对待测样本进行裂解；
S40、通过旋转阀的旋转控制将结合液吸入注塞器，通过结合液中的核酸捕获磁珠吸附裂解后的待测样本中的核酸；
S50、通过旋转阀的旋转控制连通注塞器腔和清洗液I腔，将清洗液I吸入注塞器，对核酸捕获磁珠进行清洗；
S60、通过旋转阀的旋转控制连通注塞器腔和清洗液II腔，将清洗液II吸入注塞器，对核酸捕获磁珠进行再次清洗；
S70、通过旋转阀的旋转控制连通注塞器腔和洗脱液腔，将洗脱液吸入注塞器，对核酸捕获磁珠中的核酸进行洗脱；
S80、通过旋转阀的旋转控制连通注塞器腔和冻干试剂腔，将包含核酸的洗脱液与冻干试剂混合，配制成PCR反应液，然后将PCR反应液全部吸入注塞器腔；
S90、通过旋转阀的旋转控制将PCR反应液分配至PCR反应腔，进行PCR反应，对核酸进行扩增；
S100、DNA探针对扩增后的核酸进行捕获，并通过检测电极输出检测结果。
本发明提供的方法，基于上述的集成全封闭式核酸检测装置。因为检测装置配备了所需的核酸提取和检测试剂，样本添加后，实现全封闭自动话处理，实现了“样本进结果出”；同时兼具极好的密闭性，这是一种全集成的核酸检测微流控芯片，可对流体进行精确的操控，密闭性非常好，使用过程中不予外部空气连通，能极大地避免外部环境对检测结果的影响，也能保护检测人员的健康。本发明的方法可实现全封闭式核酸提取及PCR检测，从样本放置完成后，就在完全封闭的状态实现样本裂解、核酸结合、清洗、洗脱以及PCR检测，保证了核酸提取及扩张过程的密闭性，减少外界气溶胶对检测结果的干扰，以及防止样本对检测人员的传染危害，实现了样本进结果出的全自动化检测过程。
下面通过具体实施例对本发明一种集成全封闭式核酸检测系统以及检测装置做进一步的解释说明。
实施例1
图6所示为本实施例提供的其中一种集成全封闭式核酸检测装置，包括样本口，用于待测样本的加入；混合池，用于进行相应溶液的混合反应；注塞器，用于抽取并混合不同的液体；旋转阀，控制各个腔室的连通；放置裂解液、清洗液I、清洗液II、洗脱液以及冻干试剂对应的腔室；废液腔，内置吸水棉；PCR反应腔，进行待测样本核酸的PCR反应；以及检测电极。
本实施例中集成全封闭式核酸检测装置的工作原理和过程如下：
1、样本前处理
A)裂解样本：
将液化后的样本加入到样本口后，盖上密封盖，将旋转阀开关转到样本口底部的接口后，连通样本口和注塞器腔，利用注塞器抽取100-1000μl的样本体积，通过流道内置的过滤膜，去除样本中的杂质。下一步，旋转阀开关转到裂解液腔，连通裂解液腔和注塞器腔，再抽取100-1000μl裂解液，并利用来回抽吸的方式，实现样本和裂解液的快速混合，等待1-15min的反应时间。
本发明适合多种临床样本，如尿液、血液、鼻咽拭子、痰液、组织、粪便等样本，可以根据具体的样本调整试剂配方及增减处理步骤。
B)核酸结合：
待样本裂解完全后，将液体完全抽入注塞器腔，然后将旋转阀转到混合池，连通混合池和注塞器腔，抽入等体积的结合液(里面包含捕获核酸的磁珠)，利用注射器的抽吸使液体混合均匀，混合时间为1-15min。
C)核酸清洗：
待磁珠完全吸附核酸后，在注塞器腔底部放入强电磁铁，将磁珠固定在注塞器腔的底部，然后将旋转阀转到废液腔，连通废液腔和注塞器腔，推动注塞器挤压液体进入废液腔(内置高强力吸水棉)。液体被排出后，将旋转阀转到清洗液I腔，连通清洗液I腔和注塞器腔，抽入100-2000μl清洗液I，利用注塞器来回抽吸充分混匀1-10min，在注塞器腔底部放入强电磁铁，将磁珠固定在注塞器腔的底部，将旋转阀转到废液腔，连通废液腔和注塞器腔，推动注塞器挤压液体进入废液腔。液体被排出后，将旋转阀转到清洗液II腔，连通清洗液II腔和注塞器腔，抽入100-2000μl清洗液II，利用注塞器来回抽吸充分混匀1-10min。
D)核酸洗脱：
待清洗完毕后，在注塞器腔底部放入强电磁铁，将磁珠固定在注塞器腔的底部，然后将旋转阀转到废液腔，连通废液腔和注塞器腔，推动注塞器挤压液体进入废液腔。液体被排出后，将旋转阀转到洗脱液腔，连通洗脱液和注塞器腔，抽入50-1000μl的洗脱液，利用注塞器来回抽吸充分混匀1-10min。
2、PCR扩增
A)配制PCR反应液
核酸洗脱后，将强电磁铁放入注塞器腔底部，将磁珠固定在注塞器底部，然后将旋转阀转到冻干粉腔，连通冻干粉腔和注塞器腔，推入固定体积的洗脱液进入冻干粉腔后，将旋转阀转到洗脱液腔，连通洗脱液腔和注塞器腔，把剩余液体全部推入洗脱液腔备用。再将旋转阀转到冻干粉腔，连通冻干粉腔和注塞器腔，利用注塞器来回抽吸使PCR反应液混合均匀，最后全部吸入注塞器腔。
B)分配PCR反应液
完成PCR反应液的配制后，将旋转阀转到PCR流道孔，连通PCR流道和注塞器腔，缓慢推动注塞器，PCR混合液沿着流道进入PCR腔。PCR腔和流道是没有沟道直接连接的，在制作PCR腔的时候，采用单面压力敏感胶作为封接层，在施压封接的时候，液体出入孔和PCR腔之间的连线不施加压力，而其它非流路区域完全密封，使流路区域处于非封接状态，当有液体从PCR混合液入口进入时，可以从未封接流路区域进入PCR腔，再从PCR腔经未封接流路区域进入废液流路出口，多余的液体最后进入到废液腔。
C)PCR扩增
待PCR混合液完成进样后，在外部压合部件的作用下，PCR腔四周单面压力敏感胶受到压力，实现PCR腔密封，工作电极表面提前接好了对应检测目标物的探针，探针的一端通过AU-S键接在电极表面，另一端接二茂铁。在外部仪器的控制下进行PCR扩增反应，该扩增反应可以是PCR、LAMP、SDA或RPA等。
3、电化学检测
修饰有巯基和二茂铁的DNA探针通过Au-S键在金电极表面自组装形成均匀分布的目标DNA捕获区，由于DNA探针上的二茂铁与金电极表面距离较近从而产生一个相对较大的峰电流信号。当加入目标DNA后，DNA探针与目标DNA杂化形成刚度较大的DNA双链，从而使DNA探针尾端的二茂铁远离金电极表面，使得方波伏安法中的峰电流减小，构建“turn-off”型电化学传感器。
综上所述，本发明提供了一种集成全封闭式核酸检测系统以及检测装置，所述系统包括了样本前处理模块、核酸扩增模块、电化学检测模块以及旋转阀。本发明的装置集成了样本采集口、过滤膜、流体控制旋转阀、密闭性注塞器等，可以对复杂样本从前处理到检测实现全集成，节省检测的前处理时间。本发明可实 现全封闭式核酸提取及PCR检测，从样本放置完成后，就在完全封闭的状态实现样本裂解、核酸结合、清洗、洗脱以及PCR检测，保证了核酸提取及扩张过程的密闭性，减少外界气溶胶对检测结果的干扰，以及防止样本对检测人员的传染危害，实现了样本进结果出的全自动化检测过程。同时，本发明采用高灵敏的电化学检测，不需要复杂的光学检测模块，而且还可以更加灵敏的实现对目标物的检测。电极表面采用特殊的电镀工艺形成圣诞树纳米结构，极大地增加了比表面积，提高了电极对目标物质的灵敏度。不仅如此，本发明通过集成旋转阀的设计，极大的缩减流路开关控制阀门的设置，简化了芯片的结构，降低了芯片的需求面积，从而缩减了生产成本。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (10)

  1. 一种集成全封闭式核酸检测系统，其特征在于，包括：
    样本前处理模块，用于提取待测样本中的核酸，所述样本前处理模块包括至少一个样本裂解单元，至少一个核酸结合单元，至少一个核酸洗脱单元；
    与所述样本前处理模块连接的核酸扩增模块，用于对提取得到的待测样本中的核酸进行扩增，所述核酸扩增模块包括至少一个体系配制单元，至少一个核酸扩增单元；
    与所述核酸扩增模块连接的电化学检测模块，用于对扩增后的核酸进行检测，所述电化学检测模块包括检测电极以及针对待测样本的DNA探针；
    样本后处理模块，与所述样本前处理模块、所述核酸扩增模块以及所述电化学检测模块分别连接，用于接收各个模块产生的废液；
    以及多个传递单元，分别设置在所述样本前处理模块与所述核酸扩增模块之间，所述核酸扩增模块与所述电化学检测模块之间，所述样本后处理模块与所述样本前处理模块之间、所述核酸扩增模块以及所述电化学检测模块之间，所述样本前处理模块中样本裂解单元、核酸结合单元以及核酸洗脱单元之间，所述核酸扩增模块中体系配制单元与核酸扩增单元之间。
  2. 根据权利要求1所述的集成全封闭式核酸检测系统，其特征在于，所述核酸检测系统还包括旋转阀，所述旋转阀控制所述核酸检测系统的传递单元的连接。
  3. 根据权利要求1所述的集成全封闭式核酸检测系统，其特征在于，所述核酸检测系统还包括计算机控制模块，用于对样本前处理模块、核酸扩增模块、电化学检测模块以及样本后处理模块分别进行连接与控制。
  4. 一种集成全封闭式核酸检测装置，其特征在于，所述核酸检测装置包括壳体以及安装在壳体内的三层结构层，所述结构层包括主体层、流道层、电极层，不同结构层之间使用胶粘或超声键合的方式实现封接；
    所述主体层包括用于样本前处理的若干腔室，其中预置用于样本前处理的试剂；还包括用于核酸扩增的冻干试剂腔，其中预置用于核酸扩增的冻干粉试剂球；还包括注塞器腔，用于放置注塞器，所述注塞器用于抽吸试剂；
    所述流道层包括用于样本前处理的若干通路，用于样本后处理的若干通路；
    所述电极层包括用于核酸扩增的PCR反应区，用于电化学检测的检测电极以及针对待测样本的DNA探针；
    所述核酸检测装置还包括旋转阀。
  5. 根据权利要求4所述的集成全封闭式核酸检测装置，其特征在于，所述主体层采用塑料注塑成型；所述用于样本前处理的若干腔室包括样本口、混合池、裂解液腔、清洗液I腔、清洗液II腔以及洗脱液腔；预封装后的试剂单独封装后放置在主体层的对应腔室位置，冻干粉试剂球放置在冻干试剂腔。
  6. 根据权利要求4所述的集成全封闭式核酸检测装置，其特征在于，所述流道层采用塑料注塑成型；所述用于样本前处理的若干通路包括样本口通路、混合池通路、裂解液通路、清洗液I通路、清洗液II通路以及洗脱液通路；所述用于样本后处理的若干通路包括若干废液通路；所述流道层还包括注塞器通路；所述流道层上表面与主体层的腔室连通，流道结构在下表面成型，下表面使用单面胶进行封闭。
  7. 根据权利要求4所述的集成全封闭式核酸检测装置，其特征在于，所述用于核酸扩增的PCR反应区包括PCR反应腔、PCR混合液入口以及PCR混合液出口。
  8. 根据权利要求4所述的集成全封闭式核酸检测装置，其特征在于，所述检测电极包括对电极、参考电极以及工作电极。
  9. 根据权利要求8所述的集成全封闭式核酸检测装置，其特征在于，所述DNA探针预设在所述工作电极表面，DNA探针的一端接在工作电极表面，另一端连接二茂铁；所述DNA探针通过Au-S键在工作电极表面自组装形成均匀分布的目标DNA捕获区。
  10. 一种如权利要求4-9任一所述的集成全封闭式核酸检测装置的检测方法，其特征在于，包括步骤：
    对待测样本进行液化，并将液化后的待测样本加入到样本口；
    通过旋转阀的旋转控制连通样本口和注塞器腔，将待测样本吸入注塞器；
    通过旋转阀的旋转控制连通注塞器腔和裂解液腔，将裂解液吸入注塞器中和待测样本混合，对待测样本进行裂解；
    通过旋转阀的旋转控制将结合液吸入注塞器，通过结合液中的核酸捕获磁珠吸附裂解后的待测样本中的核酸；
    通过旋转阀的旋转控制连通注塞器腔和清洗液I腔，将清洗液I吸入注塞器，对核酸捕获磁珠进行清洗；
    通过旋转阀的旋转控制连通注塞器腔和清洗液II腔，将清洗液II吸入注塞器，对核酸捕获磁珠进行再次清洗；
    通过旋转阀的旋转控制连通注塞器腔和洗脱液腔，将洗脱液吸入注塞器，对核酸捕获磁珠中的核酸进行洗脱；
    通过旋转阀的旋转控制连通注塞器腔和冻干试剂腔，将包含核酸的洗脱液与冻干试剂混合，配制成PCR反应液，然后将PCR反应液全部吸入注塞器腔；
    通过旋转阀的旋转控制将PCR反应液分配至PCR反应腔，进行PCR反应，对核酸进行扩增；
    DNA探针对扩增后的核酸进行捕获，并通过检测电极输出检测结果。
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