CN103923817B - A kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR - Google Patents
A kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR Download PDFInfo
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Abstract
The present invention relates to a kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR, this system comprises anti-corrosion alloy material structure, injection plunger, stepper-motor, waste liquid microchannel, leading screw, described leading screw is provided with slide block, slide block is reaction cavity, and magnetic bionanoparticles is placed in this slide block central authorities; The present invention adopts linear pattern reaction structure, and the reagent that avoiding works repeatedly brings mixes, and improves purity and the concentration of final DNA, and reaction is more rapidly smooth, facilitates disposable reagent filling.The present invention can adopt singlechip microprocessor integrated control system, has both decreased unnecessary peripherals, makes system more microminiaturized; And function i ntegration, makes stepper-motor and injection plunger cooperatively carry out the dynamically working of completion system, improves cut-and-try work efficiency.
Description
Technical field
The present invention relates to a kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR, belong to biology and analytical chemistry and medical science.
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 growing normally organism, grow and having a very important role in the vital movement such as breeding, 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 the 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 these shortcomings well, realize sample quick, efficiently prepare, be following nucleic acid purification method development an important directions.Paramagnetic particle method do not need centrifugal, do not need to add plurality of reagents, simple to operate, meet nucleic acid automatization extract requirement, be following nucleic acid purification method development an important directions.And on this basis, nucleic acid extraction technology is combined with real-time PCR detection technology, whole biological extraction Monitoring systems can be made integrated.
The process that paramagnetic particle method extracts nucleic acid is by cell pyrolysis liquid lysing cell, and from cell, free nucleic acid molecule is out adsorbed onto magnetic-particle surface by special, and the impurity such as protein are not stayed in the solution by adsorbing.After reaction certain hour, then under the action of a magnetic field, magnetic-particle is separated with liquid, reclaim particle, more namely can obtain pure DNA with elution, namely process is " cracking-combination-washing-wash-out " four steps.
Cell pyrolysis liquid is wherein a kind of protein denaturant, vegeto-animal lysis can be made, and make the protein denaturation that is combined with DNA, the free release of DNA, magnetic ball can adsorption of DNA specifically, by washing, removes the impurity such as RNA, protein beyond DNA, again because of the DNA of elutriant Dissociative on magnetic bead, finally obtain purity and all very high DNA of 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, under the effect of outside magnetic field can displacement and concentrate, after removing foreign field, a little vibration or suction can be dispersed in again in liquid, thus the separation of solid liquid phase is become very efficient and convenient, the very high targeting substance of purity can be obtained by simple wash-out.The advantage of paramagnetic particle method is: whole system automated operation can ensure that experimental result is stablized, the difference avoiding manual operation to cause and mistake; Intellectualized operation system, pollutes between strict control punch hole, adopts disposable plastic to extract sleeve pipe, avoids between different batches and pollute.
Therefore linear pattern DNA extraction system is in order to avoid the centrifugation step in traditional method for extracting nucleic acid, chooses paramagnetic particle method and extracts nucleic acid.Because paramagnetic particle method only needs to complete whole leaching process with reagent, so can the volume of micro whole system greatly, and shorten the reaction times, improve running efficiency of system, thus meet the following portability needs extracting detection system.In addition paramagnetic particle method can extract particular target DNA targetedly, decreases the impurity of reaction result, the error that after reducing, 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 detected towards PCR, it is mainly applicable to based on the DNA extraction work of microstructure to target sample, and can be subsequently applied to real-time PCR detection work; Simultaneously the present invention, by micro-fluidic DNA extraction module and discharging of waste liquid module two module integrations, realizes portable minisize DNA extraction system, reaches that function i ntegration, structure are miniature, the target of the little full-automatic extraction of lightweight.
To achieve these goals, the technical solution used in the present invention is a kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR, this system comprises anti-corrosion alloy material structure, injection plunger, stepper-motor, waste liquid microchannel, leading screw, described leading screw is provided with slide block, slide block is reaction cavity, and magnetic bionanoparticles is placed in this slide block central authorities; Specifically, microchannel containing eight band injection plungers in described anti-corrosion alloy material structure, the microchannel of these eight band injection plungers is arranged on anti-corrosion alloy material scantling length direction, and the microchannel of these eight band injection plungers forms the alternately module of whole reagent; Described stepper-motor and leading screw are arranged on anti-corrosion alloy material inside configuration, leading screw is equipped with slide block, this slide block can move along leading screw direction and two ends have through hole, described clear size of opening and each microchannel measure-alike, leading screw is with the axis direction of the microchannel of injection plunger mutually vertical with eight, stepper-motor and leading screw construction system power plant module; Be with the microchannel of injection plunger symmetrical with leading screw with eight, anti-corrosion alloy material structure opposite side is provided with waste liquid microchannel, described anti-corrosion alloy material structure and waste liquid microchannel construction system devil liquor recovery module.
Compared with existing DNA extraction technology, the present invention has following beneficial effect.
1, the present invention is compared to traditional DNA extraction method, as dense salt method, anionic detergent method, phenol extraction method etc., does not have centrifugation step, has both simplified the complicacy of system, reduce volume again.System architecture adds Automatic control of single chip microcomputer again, saves the working hour, makes over all Integration, does basis for follow-up with the combination of 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 present system simplifies volume, get micro-specific objective sample and extract, improve whole work efficiency and portability.
3, the present invention adopts linear pattern reaction structure, and the reagent that avoiding works repeatedly brings mixes, and improves purity and the concentration of final DNA, and reaction is more rapidly smooth, facilitates disposable reagent filling.
4, the present invention can adopt singlechip microprocessor integrated control system, has both decreased unnecessary peripherals, makes system more microminiaturized; And function i ntegration, makes stepper-motor and injection plunger cooperatively carry out the dynamically working of completion system, improves cut-and-try work efficiency.
Accompanying drawing explanation
Fig. 1 is linear pattern micro-fluidic DNA extraction system summary 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, magnetic bionanoparticles, 2-1, first channel, 2-2, second passage, 2-3, third channel, 2-4, four-way, 2-5, Five-channel, 2-6, Hexamermis spp, 2-7, the 7th passage, 2-8, the 8th passage.
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 detected towards PCR, 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 5 is provided with slide block, slide block is reaction cavity, and magnetic bionanoparticles 6 is placed in this slide block central authorities; Specifically, microchannel containing eight band injection plungers 2 in described anti-corrosion alloy material structure 1, 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; It is inner that described stepper-motor 3 and leading screw 5 are arranged on anti-corrosion alloy material structure 1, leading screw 5 is equipped with slide block, this slide block can move along leading screw 5 direction and two ends have through hole, described clear size of opening and each microchannel measure-alike, leading screw 5 is with the axis direction of the microchannel of injection plunger 2 mutually vertical with eight, stepper-motor 3 and leading screw 5 construction system power plant module; Be with the microchannel of injection plunger 2 symmetrical with leading screw 5 with eight, anti-corrosion alloy material structure 1 opposite side is provided with waste liquid microchannel 4, described anti-corrosion alloy material structure 1 and waste liquid microchannel 4 construction system devil liquor recovery module.
Eight in anti-corrosion alloy material structure 1 wherein containing being include four kinds of different biological reagents in eight microchannels of injection plunger 2.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 eight microchannels of first channel 2-1 to the 8th passage 2-8 respectively.Eight microchannels are included as respectively from top to bottom: be be be all be elutriant in washings, the 8th passage 2-8 in salt washing lotion, four-way 2-4 to the 7th passage 2-7 in cell pyrolysis liquid, third channel 2-3 in reactant stoste, second passage 2-2 in first channel 2-1; Stepper-motor 3 controls leading screw 5 pairs of injection plungers 2 and carries out time segment propelling, is pushed by the reagent in eight passages and entreats in systems in which in the slide block containing magnetic bionanoparticles 6 on leading screw 5 and reaction chamber, carried out whole biological respinse.
Whole reaction principle is: first by the extruding of injection plunger 2, reactant stoste in first channel 2-1 is transported to central authorities and carries out initial reaction containing in the slide block of magnetic bionanoparticles 6, promote slide block by stepper-motor 3 rotational lead screw 5 afterwards to advance, make aiming at of slide block and second passage 2-2; Then by the extruding of injection plunger 2, cell pyrolysis liquid in second passage 2-2 is transported in central slide block and reacts with reactant, make vegeto-animal cell membrane lysis and the protein denaturation be combined with DNA, in such reactant, target dna is dissociated release, and at this moment target dna can adsorb by magnetic bionanoparticles 6; With the principle of work of above-mentioned stepper-motor and leading screw, central slide block is aimed at successively the porthole of third channel 2-3 to the 8th 2-8, by the extruding of the injection plunger 2 in each passage, corresponding reagent is pushed central slide block and reacts.Essence is first cleaned by the salt washing lotion in passage 2-3 the DNA of thick extraction before, remove residual solubilized salt in DNA, washed by the washings in four passages of four-way 2-4 to the 7th passage 2-7 again, remove the impurity such as RNA, protein beyond DNA, dissociate finally by the elutriant in the 8th passage 2-8, the DNA be adsorbed on magnetic bionanoparticles 6 is separated, obtains purity and all very high DNA of concentration.
Such as shown in Fig. 1 linear pattern micro-fluidic DNA extraction system summary composition vertical view, the structure on the right is the drainage system of 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 reacted each time is discharged from central slide block, facilitates waste collection, avoid wasting resource, contaminate environment.
Claims (3)
1. the micro-fluidic DNA extraction system of linear pattern detected 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), (5) are provided with slide block with described leading screw, slide block is reaction cavity, and magnetic bionanoparticles (6) is placed in this slide block central authorities; Specifically, microchannel containing eight bands injection plunger (2) in described anti-corrosion alloy material structure (1), the microchannel of these eight bands injection plunger (2) is arranged on anti-corrosion alloy material structure (1) length direction, and the microchannel of these eight bands injection plunger (2) forms the alternately module of whole reagent; It is inner that described stepper-motor (3) and leading screw (5) are arranged on anti-corrosion alloy material structure (1), (5) are equipped with slide block with leading screw, this slide block is along the motion of leading screw (5) direction and two ends have through hole, described clear size of opening and each microchannel measure-alike, leading screw (5) is with the axis direction of the microchannel of injection plunger (2) mutually vertical with eight, stepper-motor (3) and leading screw (5) construction system power plant module; Be with the microchannel of injection plunger (2) symmetrical with leading screw (5) with eight, anti-corrosion alloy material structure (1) opposite side is provided with waste liquid microchannel (4), described anti-corrosion alloy material structure (1) and waste liquid microchannel (4) construction system devil liquor recovery module;
Eight in anti-corrosion alloy material structure (1) wherein containing being include four kinds of different biological reagents in eight microchannels of injection plunger (2); In anti-corrosion alloy material structure (1), include eight microchannels of first channel (2-1) to the 8th passage (2-8) respectively; Eight microchannels are included as in first channel (2-1) respectively is from top to bottom be be all be elutriant in washings, the 8th passage (2-8) in salt washing lotion, four-way (2-4) to the 7th passage (2-7) in cell pyrolysis liquid, third channel (2-3) in reactant stoste, second passage (2-2); Stepper-motor (3) controls leading screw (5) and carries out time segment propelling to injection plunger (2), reagent in eight passages is pushed entreat in systems in which on leading screw (5) containing in the slide block of magnetic bionanoparticles (6) and reaction chamber, carried out whole biological respinse.
2. a kind of micro-fluidic DNA extraction system of linear pattern detected 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 to central authorities and carries out initial reaction containing in the slide block of magnetic bionanoparticles (6), promote slide block by stepper-motor (3) rotational lead screw (5) afterwards to advance, make aiming at of slide block and second passage (2-2); Then by the extruding of injection plunger (2), cell pyrolysis liquid in second passage (2-2) is transported in central slide block and reacts with reactant, make vegeto-animal cell membrane lysis and the protein denaturation be combined with DNA, in such reactant, target dna is dissociated release, and at this moment target dna adsorbs by magnetic bionanoparticles (6); With the principle of work of above-mentioned stepper-motor and leading screw, by central slide block successively aligned with channel third channel (2-3) to the porthole of the 8th passage (2-8), by the extruding of the injection plunger (2) in each passage, corresponding reagent is pushed central slide block and reacts; Essence is first cleaned by the salt washing lotion in third channel (2-3) the DNA of thick extraction before, remove residual solubilized salt in DNA, washed to the washings in four passages of the 7th passage (2-7) by four-way (2-4) again, remove RNA, the protein impurities beyond DNA, dissociate finally by the elutriant in the 8th passage (2-8), the DNA be adsorbed on magnetic bionanoparticles (6) is separated, obtains purity and all very high DNA of concentration.
3. a kind of micro-fluidic DNA extraction system of linear pattern detected towards PCR according to claim 1, is characterized in that: in whole drainage system passage, has thieving paper to carry out waste liquid guiding.
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