CN103923817A - PCR detection-oriented linear microfluidic DNA extraction system - Google Patents
PCR detection-oriented linear microfluidic DNA extraction system Download PDFInfo
- Publication number
- CN103923817A CN103923817A CN201410143922.8A CN201410143922A CN103923817A CN 103923817 A CN103923817 A CN 103923817A CN 201410143922 A CN201410143922 A CN 201410143922A CN 103923817 A CN103923817 A CN 103923817A
- Authority
- CN
- China
- Prior art keywords
- slide block
- passage
- dna
- leading screw
- microchannel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention relates to a PCR detection-oriented linear microfluidic DNA extraction system. The system comprises a corrosion-resistant alloy material structure, an injection piston, a stepper motor, a liquid waste microchannel and a screw, wherein a slide block is arranged on the screw; the slide block is a reaction cavity; biological nanometer magnetic beads are arranged at the center of the slide block. The linear reaction structure is adopted, mixing of reagents caused by repeated work is avoided, the final purity and the concentration of DNA are improved, the reaction is smoother and faster, and one-time bulking of the reagent is facilitated. A single chip microprocessor integrated control system can be adopted, excessive peripheral equipment is reduced, the system is more miniaturized, and the function is integrated, so that the stepper motor and the injection piston finish power work of the system in common, and the experimental work efficiency is improved.
Description
Technical field
The present invention relates to a kind of micro-fluidic DNA extraction system of linear pattern detecting towards PCR, belong to biology and analytical chemistry and medical science detection field.
Background technology
Nucleic acids research is the important topic in modern biology, medical research.Nucleic acid is as the carrier of genetic information, it is the basic substance of genetic expression, except grow normally, grow organism and the vital movement such as breeding in have a very important role, the abnormal conditions of it and life, as tumour generation, radiotherapy damage etc. also have substantial connection.Therefore nucleic acid is the important topic in modern biology, medical research.The processes such as traditional separate nucleic acid technology comprises precipitation, centrifugal, numerous and diverse, the time-consuming length of step of these purification process, yield are low, and contact toxic reagent, is difficult to realize automated operation; And adopt magnetic carrier microballoon isolation technique just can overcome well these shortcomings, and realize fast, efficiently preparing of sample, be an important directions of following nucleic acid purification method development.That paramagnetic particle method does not need is centrifugal, do not need to add plurality of reagents, simple to operate, meets nucleic acid automatization and extracts requirement, is an important directions of following nucleic acid purification method development.And on this basis, nucleic acid extraction technology is combined with real-time fluorescence PCR detection technique, can make whole biological extraction Monitoring systems integrated.
It is by cell pyrolysis liquid lysing cell that paramagnetic particle method extracts the process of nucleic acid, and from cell, free nucleic acid molecule is out by the special magnetic-particle surface that is adsorbed onto, and the impurity such as protein are not adsorbed and stay in solution.After reaction certain hour, then under the action of a magnetic field, magnetic-particle and liquid are separated, recovery particle, more can obtain pure DNA with elutriant wash-out, process is " cracking-combination-washing-wash-out " four steps.
Cell pyrolysis liquid is wherein a kind of protein denaturant, can make vegeto-animal lysis, and make the protein denaturation of being combined with DNA, DNA is free to be discharged, magnetic ball adsorption of DNA specifically, by washing, removes the DNA impurity such as RNA, protein in addition, again because of the DNA of elutriant Dissociative on magnetic bead, finally obtain all very high DNA of purity and concentration.Biomagnetic beads is wherein a kind of novel functionalization solid carrier, its pan coating has active group, can with various bioactivators generation coupling, with the feature such as mobility and solid magnetic material of liquid, can displacement under the effect of outside magnetic field and concentrate, when removing after foreign field, vibration or suction can be dispersed in again in liquid a little, it is very efficient and convenient that thereby the separation that makes solid liquid phase becomes, and can obtain by simple wash-out the targeting substance that purity is very high.The advantage of paramagnetic particle method is: whole system can automated operation ensure that experimental result is stable, difference and the mistake of avoiding manual operation to cause; Intellectualized operation system, strict control punch pollutes between hole, adopts disposable plastic to extract sleeve pipe, has avoided polluting between different batches.
Therefore linear pattern DNA extraction system, for fear of the centrifugation step in traditional method for extracting nucleic acid, is chosen paramagnetic particle method and is extracted nucleic acid.Because paramagnetic particle method only need to complete whole leaching process with reagent, thus the volume of micro whole system greatly, and shortened the reaction times, improve running efficiency of system, thereby met the following portability needs that extract detection system.In addition paramagnetic particle method can extract special target dna targetedly, has reduced the impurity of reaction result, the error that after having reduced, real-time pcr fluorescence detects.
Summary of the invention
The object of the present invention is to provide a kind of micro-fluidic DNA extraction system of linear pattern detecting towards PCR, it is mainly applicable to the DNA extraction work to target sample based on microstructure, and can be subsequently applied to real-time fluorescence PCR testing; Simultaneously the present invention, by micro-fluidic DNA extraction module and two module integrations of discharging of waste liquid module, realizes portable minisize DNA extraction system, reaches that function is integrated, structure is miniature, the target of the little full-automatic extraction of lightweight volume.
To achieve these goals, the technical solution used in the present invention is a kind of micro-fluidic DNA extraction system of linear pattern detecting towards PCR, this system comprises anti-corrosion alloy material structure, injection plunger, stepper-motor, waste liquid microchannel, leading screw, on described leading screw, slide block is installed, slide block is reaction cavity, and biological nano magnetic bead is placed in this slide block central authorities; Particularly, in described anti-corrosion alloy material structure, contain eight microchannels with injection plunger, these eight microchannels with injection plunger are arranged on anti-corrosion alloy material scantling length direction, and these eight microchannels with injection plunger form the alternately module of whole reagent; Described stepper-motor and leading screw are arranged on anti-corrosion alloy material inside configuration, slide block is housed on leading screw, this slide block can move and two ends have through hole along leading screw direction, described clear size of opening and each microchannel are measure-alike, leading screw is mutually vertical with the axis direction of eight microchannels with injection plunger, stepper-motor and leading screw construction system power plant module; Symmetrical with leading screw with eight microchannels with injection plunger, be provided with waste liquid microchannel at anti-corrosion alloy material structure opposite side, described anti-corrosion alloy material structure and waste liquid microchannel construction system waste liquid recycling module.
Compared with existing DNA extraction technology, the present invention has following beneficial effect.
1, the present invention, compared to traditional DNA extraction method, as dense salt method, anionic detergent method, phenol extraction process etc., does not have centrifugation step, has both simplified the complicacy of system, has dwindled again volume.System architecture adds again Automatic control of single chip microcomputer, has saved the working hour, makes over all Integration, does basis for follow-up with combination PCR fluorescence detecting system.In addition traditional DNA extraction method, the harmful and contaminate environment of reagent; And the present invention adopts reagent environmental protection more, and there is liquid waste treatment system, saving resource.
2, the present invention contrasts existing paramagnetic particle method DNA extraction instrument, and existing paramagnetic particle method extracts instrument and once extracts the working hour more than one hour, bulky; And system simplification of the present invention volume, get micro-specific objective sample to extract, improved whole work efficiency with portable.
3, the present invention adopts linear pattern reaction structure, and the reagent bringing of having avoided repeatedly working mixes, and has improved purity and the concentration of final DNA, and reaction is more smooth rapidly, facilitates disposable reagent filling.
4, the present invention can adopt singlechip microprocessor integrated control system, has both reduced unnecessary peripherals, makes system more microminiaturized; And function is integrated, make stepper-motor jointly carry out the dynamically working of completion system together with injection plunger, improve cut-and-try work efficiency.
Brief description of the drawings
Fig. 1 is the micro-fluidic DNA extraction system summary of linear pattern composition vertical view.
Fig. 2 is that figure is cutd open in the side of the micro-fluidic DNA extraction system of linear pattern.
In figure: 1, anti-corrosion alloy material structure; 2, injection plunger; 3, stepper-motor; 4, waste liquid microchannel; 5, leading screw; 6, biological nano magnetic bead.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
As Fig. 1-2, a kind of micro-fluidic DNA extraction system of linear pattern detecting towards PCR, this system comprises anti-corrosion alloy material structure 1, injection plunger 2, stepper-motor 3, waste liquid microchannel 4, leading screw 5, on described leading screw 5, slide block is installed, slide block is reaction cavity, and biological nano magnetic bead 6 is placed in this slide block central authorities; Particularly, in described anti-corrosion alloy material structure 1, contain eight microchannels with injection plunger 2, these eight microchannels with injection plunger 2 are arranged on anti-corrosion alloy material structure 1 length direction, and these eight microchannels with injection plunger 2 form the alternately module of whole reagent; Described stepper-motor 3 is arranged on anti-corrosion alloy material structure 1 inside with leading screw 5, on leading screw 5, slide block is housed, this slide block can move and two ends have through hole along leading screw 5 directions, described clear size of opening and each microchannel are measure-alike, leading screw 5 is mutually vertical with the axis direction of eight microchannels with injection plunger 2, stepper-motor 3 and leading screw 5 construction system power plant modules; Symmetrical with leading screw 5 with eight microchannels with injection plunger 2, be provided with waste liquid microchannel 4 at anti-corrosion alloy material structure 1 opposite side, described anti-corrosion alloy material structure 1 and waste liquid microchannel 4 construction system waste liquid recycling modules.
It is to include four kinds of different biological reagents that in anti-corrosion alloy material structure 1 eight are wherein contained in eight microchannels of injection plunger 2.As shown in DNA extraction system summary composition vertical view as micro-fluidic in Fig. 1 linear pattern, in anti-corrosion alloy material structure 1, include respectively eight microchannels of 2-1 to 2-8.Eight microchannels are included as respectively from top to bottom: in passage 2-1, be in reactant stoste, passage 2-2, to be for salt washing lotion, passage 2-4 are to be elutriant in washings, passage 2-8 to passage 2-7 in cell pyrolysis liquid, passage 2-3; Stepper-motor 3 is controlled leading screw 5 injection plunger 2 is carried out to time segment propelling, and the slide block that contains biological nano magnetic bead 6 that the reagent in eight passages is pushed on system central authorities leading screw 5 is in reaction chamber, completes whole biological respinse.
Whole reaction principle is: first by the extruding of injection plunger 2, reactant stoste in passage 2-1 is transported in the slide block that central authorities contain biological nano magnetic bead 6 and carries out initial reaction, promote slide block by stepper-motor 3 rotational lead screws 5 afterwards and advance, make aiming at of slide block and passage 2-2; Then by the extruding of injection plunger 2, cell pyrolysis liquid in passage 2-2 is transported in central slide block and is reacted with reactant, the protein denaturation that makes vegeto-animal cytolemma cracking and be combined with DNA, the target dna release that dissociated in reactant like this, at this moment biological nano magnetic bead 6 can be by target dna absorption; With the principle of work of above-mentioned stepper-motor and leading screw, by the central slide block successively porthole of aligned with channel 2-3 to 2-8, by the extruding of the injection plunger 2 in each passage, corresponding reagent is pushed to central slide block and react.Essence is that the DNA of thick extraction before is first cleaned by the salt washing lotion in passage 2-3, remove residual solubilized salt in DNA, wash to four washingss of passage 2-7 by passage 2-4 again, remove the DNA impurity such as RNA, protein in addition, finally dissociate by the elutriant in passage 2-8, the DNA being adsorbed on biological nano magnetic bead 6 is separated, obtain all very high DNA of purity and concentration.
As shown in DNA extraction system summary composition vertical view as micro-fluidic in Fig. 1 linear pattern, the drainage system that the structure on the right is whole system in the drawings.The structure on the right and the reaction structure symmetry on the left side, reagent react each time has corresponding waste liquid apocenosis passage.In whole drainage system passage, there is thieving paper to carry out waste liquid guiding, by thieving paper and action of gravity, the waste liquid of reaction is each time discharged from central slide block, facilitate waste collection, the resource that avoids waste, contaminate environment.
Claims (3)
1. the micro-fluidic DNA extraction system of linear pattern detecting towards PCR, it is characterized in that: this system comprises anti-corrosion alloy material structure (1), injection plunger (2), stepper-motor (3), waste liquid microchannel (4), leading screw (5), described leading screw is provided with slide block on (5), slide block is reaction cavity, and biological nano magnetic bead (6) is placed in this slide block central authorities; Particularly, in described anti-corrosion alloy material structure (1), contain the microchannel of eight band injection plungers (2), the microchannel of these eight band injection plungers (2) is arranged on anti-corrosion alloy material structure (1) length direction, and the microchannel of these eight band injection plungers (2) forms the alternately module of whole reagent; Described stepper-motor (3) is arranged on anti-corrosion alloy material structure (1) inside with leading screw (5), leading screw is equipped with slide block on (5), this slide block can have through hole along the motion of leading screw (5) direction and two ends, described clear size of opening and each microchannel are measure-alike, leading screw (5) is mutually vertical with the axis direction of the microchannel of eight band injection plungers (2), stepper-motor (3) and leading screw (5) construction system power plant module; Symmetrical with leading screw (5) with the microchannel of eight band injection plungers (2), be provided with waste liquid microchannel (4) at anti-corrosion alloy material structure (1) opposite side, described anti-corrosion alloy material structure (1) and waste liquid microchannel (4) construction system waste liquid recycling module;
It is to include four kinds of different biological reagents that in anti-corrosion alloy material structure (1) eight are wherein contained in eight microchannels of injection plunger (2); In anti-corrosion alloy material structure (1), include respectively eight microchannels of 2-1 to 2-8; From top to bottom eight microchannels be included as respectively in passage 2-1 in reactant stoste, passage 2-2 in cell pyrolysis liquid, passage 2-3 for salt washing lotion, passage 2-4 are to be elutriant in washings, passage 2-8 to passage 2-7; Stepper-motor (3) is controlled leading screw (5) injection plunger (2) is carried out to time segment propelling, the slide block that contains biological nano magnetic bead (6) that reagent in eight passages is pushed on system central authorities' leading screws (5) is in reaction chamber, completes whole biological respinse.
2. a kind of micro-fluidic DNA extraction system of linear pattern detecting towards PCR according to claim 1, it is characterized in that: whole reaction principle is, first by the extruding of injection plunger (2), reactant stoste in passage (2-1) is transported in the slide block that central authorities contain biological nano magnetic bead (6) and carries out initial reaction, promote slide block by stepper-motor (3) rotational lead screw (5) afterwards and advance, make aiming at of slide block and passage 2-2; Then by the extruding of injection plunger (2), cell pyrolysis liquid in passage 2-2 is transported in central slide block and is reacted with reactant, the protein denaturation that makes vegeto-animal cytolemma cracking and be combined with DNA, the target dna release that dissociated in reactant like this, at this moment biological nano magnetic bead (6) can be by target dna absorption; With the principle of work of above-mentioned stepper-motor and leading screw, by the central slide block successively porthole of aligned with channel 2-3 to 2-8, by the extruding of the injection plunger in each passage (2), corresponding reagent is pushed to central slide block and react.Essence is that the DNA of thick extraction before is first cleaned by the salt washing lotion in passage 2-3, remove residual solubilized salt in DNA, wash to four washingss of passage 2-7 by passage 2-4 again, remove DNA RNA, protein impurities in addition, finally dissociate by the elutriant in passage 2-8, the DNA being adsorbed on biological nano magnetic bead (6) is separated, obtain all very high DNA of purity and concentration.
3. a kind of micro-fluidic DNA extraction system of linear pattern detecting towards PCR according to claim 1, is characterized in that: in whole drainage system passage, have thieving paper to carry out waste liquid guiding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410143922.8A CN103923817B (en) | 2014-04-10 | 2014-04-10 | A kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410143922.8A CN103923817B (en) | 2014-04-10 | 2014-04-10 | A kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103923817A true CN103923817A (en) | 2014-07-16 |
| CN103923817B CN103923817B (en) | 2015-10-28 |
Family
ID=51142216
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410143922.8A Expired - Fee Related CN103923817B (en) | 2014-04-10 | 2014-04-10 | A kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103923817B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232469A (en) * | 2014-09-11 | 2014-12-24 | 北京化工大学 | Magnetic bead-based sample treatment and nucleic acid automatic extraction system |
| CN113092796A (en) * | 2021-04-02 | 2021-07-09 | 浙江大学 | Accurate sample introduction system for water phase and oil phase |
| CN113913271A (en) * | 2021-07-19 | 2022-01-11 | 杭州奥盛仪器有限公司 | Gene detection kit and gene detection equipment |
| CN116679043A (en) * | 2023-08-03 | 2023-09-01 | 天津大学 | Robot suitable for magnetophoresis analysis platform, preparation method and magnetophoresis analysis platform |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070248497A1 (en) * | 2004-09-28 | 2007-10-25 | Cleveland Biosensors Pty Ltd | Microfluidic device |
| US20090176316A1 (en) * | 2004-07-28 | 2009-07-09 | Expert Services Group, Inc. | Automated fluid handling cartridge, fluid processing system, and methods |
-
2014
- 2014-04-10 CN CN201410143922.8A patent/CN103923817B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090176316A1 (en) * | 2004-07-28 | 2009-07-09 | Expert Services Group, Inc. | Automated fluid handling cartridge, fluid processing system, and methods |
| US20070248497A1 (en) * | 2004-09-28 | 2007-10-25 | Cleveland Biosensors Pty Ltd | Microfluidic device |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232469A (en) * | 2014-09-11 | 2014-12-24 | 北京化工大学 | Magnetic bead-based sample treatment and nucleic acid automatic extraction system |
| CN113092796A (en) * | 2021-04-02 | 2021-07-09 | 浙江大学 | Accurate sample introduction system for water phase and oil phase |
| CN113913271A (en) * | 2021-07-19 | 2022-01-11 | 杭州奥盛仪器有限公司 | Gene detection kit and gene detection equipment |
| CN116679043A (en) * | 2023-08-03 | 2023-09-01 | 天津大学 | Robot suitable for magnetophoresis analysis platform, preparation method and magnetophoresis analysis platform |
| CN116679043B (en) * | 2023-08-03 | 2023-11-03 | 天津大学 | Robot suitable for magnetophoresis analysis platform, preparation method and magnetophoresis analysis platform |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103923817B (en) | 2015-10-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108660074B (en) | Integrated solution for nucleic acid extraction PCR amplification detection | |
| JP6041448B2 (en) | Sample preparation device and method | |
| JP5920895B2 (en) | Method and device for isolating cells from heterogeneous solutions using microfluidic capture vortices | |
| CN112195099B (en) | Micro-fluidic chip for nucleic acid detection | |
| JP2011530084A (en) | Methods and systems for selective isolation of target biological molecules in a universal system | |
| US11905550B2 (en) | Process for preparing a biological sample | |
| CN104762193A (en) | Microfluidic device for automatic extraction of nucleic acid | |
| CN104334725A (en) | Biomolecule isolation | |
| CN103923817B (en) | A kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR | |
| CN104130932A (en) | Agarose microfluidic chip based bacteria enrichment device | |
| CN102586226B (en) | Microfluidic chip nucleic acid purification platform based on continuous liquid drop control | |
| Kim et al. | Automated microfluidic DNA/RNA extraction with both disposable and reusable components | |
| CN108070585A (en) | A kind of kit and its method based on magnetic bead technology extraction poba gene group DNA | |
| US20160001284A1 (en) | Fluidic Interfacing System and Assembly | |
| JP7301416B2 (en) | ANALYTE COLLECTION DEVICE, ANALYTE COLLECTION METHOD USING THE SAME, AND ANALYTE INSPECTION SYSTEM | |
| KR20160034694A (en) | Microfluidic apparatus for isolation, method for isolation using the same, and isolation kit for circulating rare cells using the same | |
| CN111304077B (en) | Integrated microfluidic chip for extracting nucleic acid and extraction method thereof | |
| CN113388513A (en) | Micro-fluidic chip for nucleic acid extraction, amplification and detection | |
| KR20130066293A (en) | Microfluidic chip for extracting nucleic acid, device comprising the same, and method for extracting nucleic acid using the same | |
| CN105203375A (en) | High-throughput plasma separation device and preparation method thereof | |
| CN217499275U (en) | Totally-enclosed nucleic acid detection chip | |
| EP3282005A1 (en) | Cell separation chip and method for separating cells by using same | |
| WO2013118990A1 (en) | Ultra-high-speed nucleic acid extracting apparatus and nucleic acid extracting method using same | |
| CN2552003Y (en) | Micro fluidic chip | |
| CN112570051A (en) | Microfluidic chip for sorting DNA, and apparatus and method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151028 Termination date: 20180410 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |