CN102115711B - Micro-flow control chip and nucleic acid extracting and purifying method - Google Patents
Micro-flow control chip and nucleic acid extracting and purifying method Download PDFInfo
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Abstract
The invention relates to a micro-flow control chip and a nucleic acid extracting and purifying method using same. The micro-flow control chip comprises a cell culture micro-unit, a mixing micro-unit, a nucleic acid collecting micro-unit and a micro-passageway, wherein the cell culture micro-unit is used for cultivating the generative cell and the peptic cell, feeding the peptic cell into the mixing micro-unit for splitting decomposition, feeding the cell lysis solution into the nucleic acid collecting micro-unit, and collecting and purifying nucleic acid; and the micro-passageway is connected with the cell culture micro-unit, the mixing micro-unit and the nucleic acid collecting micro-unit and is the passageway which charges and discharges the liquid. The cell culture, the cell lysis and the nucleic acid collection and the purification are integrated into one micro-flow control chip, so that the extraction and purification working procedure of the nucleic acid can be optimized, the extraction and purification efficiency can be greatly improved, the manual operation procedure can be simplified, the space-time discrimination information which is hardly realized by a conventional method can be realized, and a better nucleic acid extracting and purifying effect can be obtained.
Description
[technical field]
The present invention relates to nucleic acid extraction and purifying field, relate in particular to a kind of micro-fluidic chip and nucleic acid extraction purification process.
[background technology]
The isolation and purification technology of biomacromolecule Yeast Nucleic Acid is the basis of carrying out the research of molecular biology each side, is the gordian technique in life science and application.Along with the development and application of microflow control technique, also be developed based on the method for extracting nucleic acid of micro-fluidic chip.It is that silicon-dioxide is fixed on the chip inwall that traditional chip extracts the nucleic acid method, perhaps the matrix such as silica gel, diatomite, granulated glass sphere, organosilane particle, magnetic bead is filled in chip channel, adsorbs nucleic acid.At first adopt the cracking tissues such as denaturing agent, Proteinase K, after discharging nucleic acid, then use phenol, chloroform extraction, isopropanol precipitating obtains nucleic acid after the removal supernatant.The traditional method complex steps, length consuming time, extraction yield is low, poor repeatability.
[summary of the invention]
Given this, be necessary to provide a kind of micro-fluidic chip of efficient, stable, easy extraction purification of nucleic acid.
Simultaneously, also be necessary to provide a kind of efficient, stable, easy nucleic acid extraction purification process.
a kind of micro-fluidic chip, comprise the cell cultures micro unit, mix micro unit, nucleic acid is collected micro unit and microchannel, described cell cultures micro unit is used for cultivating propagation and peptic cell, to digest good cell passes into to described mixing micro unit again, described mixing micro unit is used for cracking and digests good cell, and cell pyrolysis liquid is passed into to described nucleic acid collection micro unit, described nucleic acid is collected micro unit and is used for collecting purification of nucleic acid, described microchannel connects described cell cultures micro unit, mix micro unit and nucleic acid and collect micro unit, and be the passage that liquid passes into and flows out.
Preferably, micro-fluidic chip is 3 tunic structures.
Preferably, 3 tunic structures comprise two-layer PDMS membrane and one deck glass substrate, and described two-layer PDMS membrane is positioned at above described glass substrate.
Preferably, this micro-fluidic chip also comprises air chamber, gas passage, horizontal little valve and vertical little valve, described air chamber is used for storage gas, the flow direction that gas passage is used for connecting air chamber and controls gas, and described horizontal little valve and vertical little valve are controlled the flow direction of liquid in described microchannel.
Preferably, air chamber, gas passage, horizontal little valve and vertical little valve are positioned between described two-layer PDMS membrane.
Preferably, among the space that the PDMS membrane in the middle of cell cultures micro unit, mixing micro unit, nucleic acid collection micro unit and microchannel are positioned at and glass substrate form.
Preferably, nucleic acid is collected micro unit and is also comprised magnetic field device, and described magnetic field device is used for the magnetic absorption ferroferric oxide magnetic nanoparticle.
Utilize the nucleic acid extraction purification process of above-mentioned micro-fluidic chip, comprise the steps:
A, cultivation contain the cell of purpose nucleic acid; B, pass into Digestive system cell is taken off wall digestion; C, pass into lysate postdigestive cell is carried out cracking, obtain containing the cell pyrolysis liquid of nucleic acid substances; D, extract purification of nucleic acid from cell pyrolysis liquid.
Preferably, also comprise carry out using successively distilled water, D-Hanks balanced salt solution that micro-fluidic chip is soaked before steps A and rinse step and ending step D after remove magnetic field, use distilled water to carry out rinse step to micro-fluidic chip.
Preferably, passing into Digestive system in step B takes off wall digestion to cell and comprises that also the phosphate buffered saline buffer that passes into before this neutral pH carries out rinse step to cell.
Preferably, step D comprises: D1, pass into the damping fluid that is suspended with ferroferric oxide magnetic nanoparticle of the acidity of pH 6.3-6.7, by the fixing ferroferric oxide magnetic nanoparticle in magnetic field; D2, will contain nucleic acid cell pyrolysis liquid by ferroferric oxide magnetic nanoparticle, under acidic conditions, nucleic acid combines formation nucleic acid-magnetic nano particle mixture and is fixed with ferroferric oxide magnetic nanoparticle; D3, pass into washings protein, the lipid cell impurity component that is not attached on ferroferric oxide magnetic nanoparticle washed; D4, the damping fluid that passes into the alkalescence of pH 8.4-8.6 carry out wash-out to nucleic acid-magnetic nano particle mixture, and under alkaline condition, nucleic acid separates with ferroferric oxide magnetic nanoparticle, collect the elutriant that contains purification of nucleic acid.
Preferably, nucleic acid is DNA.
Focus on a micro-fluidic chip by the collection purifying with the cultivation of cell, lysis, nucleic acid, optimized nucleic acid extraction purifying flow process, improved greatly the efficient of extracting purifying, simplify the manual operation program, and realized the time-space resolution information that traditional method is difficult to realize, obtained nucleic acid extraction purification effect preferably.
[description of drawings]
Fig. 1 is micro-fluidic chip two dimensional structure schematic diagram of the present invention.
Fig. 2 is the structural representation of ferroferric oxide magnetic nanoparticle.
[embodiment]
The chip body of micro-fluidic chip comprises two-layer polydimethylsiloxane (PDMS) film and one deck glass substrate, the setting of two-layer PDMS film is positioned at above glass substrate, adopt three layers of chip design, make extraction and the purifying whole process of whole nucleic acid comprise cell cultures, lysis, separate nucleic acid, wash-out and collection etc. are integrated on a chip, are suitable for the extraction and purification of multiple nucleic acids sample.
The micro-fluidic chip of 3 tunic structures, above 2 layers be the PDMS film, the below is glass substrate, is the microfluidic channel structure between middle PDMS film and glass substrate, comprise cell cultures micro unit 110, mixing micro unit 120, nucleic acid collection micro unit 130 and microchannel, as shown in Figure 1.The compartment that forms between upper two layers PDMS film comprises air chamber and gas passage, and air chamber is mainly used in storing gas, is between the buffering of gaseous matter, and gas passage is mainly used in controlling turnover and the flow direction of gas; And be integrated with horizontal little valve and vertical little valve, as horizontal little valve 151, vertical little valve 152, the turnover of controlling liquid flows to.
Cell cultures micro unit 110 is mainly used in culturing cell; Mix micro unit 120 and be mainly used in the cultured cell of cracking; Nucleic acid is collected the ferroferric oxide magnetic nanoparticle that micro unit 130 utilizes magnetic field absorption (ferroferric oxide magnetic nanoparticle and preparation method thereof description that sees below, and separate case has been applied for a patent) adsorb nucleic acid, form nucleic acid-magnetic nano particle mixture with nucleic acid, thereby reach the purpose that nucleic acid separates with the cell impurity component; The microchannel comprises first channel 141, second passage 142, third channel 143, four-way 144, Five-channel 145, the 6th passage 146, the 7th passage 147 and the 8th passage 148.
The cell suspending liquid that contains purpose nucleic acid that will be to be cultivated by leftmost first channel 141 is passed into cell cultures micro unit 110, and cell is in cell cultures micro unit 110 places cultivation propagation, and the waste liquid that produces in culturing process flows out from second passage 142; After cell cultures finishes, pass into Digestive system from first channel 141 cell is taken off wall digestion, the cell that digestion is good is transported to and mixes micro unit 120, meanwhile by third channel 143, cell pyrolysis liquid is passed into and mixes micro unit 120, the good cell of digestion is mixing micro unit 120 places by the abundant cracking of cell pyrolysis liquid; Treat that lysis becomes cell debris, after the nucleic acid such as DNA discharge from nucleus, by four-way 144, ferroferric oxide magnetic nanoparticle being passed into nucleic acid collects in micro unit 130, be distributed with magnetic field below nucleic acid collection micro unit 130, ferroferric oxide magnetic nanoparticle is fixed on substrate by magnetic field gravitation, and the damping fluid that is used for the suspension ferroferric oxide magnetic nanoparticle flows out from Five-channel 145; And then the cell pyrolysis liquid that will contain nucleic acid is passed into nucleic acid and collects micro unit 130, and in the solution of pH acidity, nucleic acid combines formation nucleic acid-magnetic nano particle mixture and is fixed on substrate with ferroferric oxide magnetic nanoparticle; Pass into acidic cleaning liquid from the 6th passage 146 nucleic acid collection micro unit 130 is washed to remove the cell impurity components such as Deproteinization, lipid, the waste liquid after washing finishes is by Five-channel 145 outflows; Pass into ealkaline buffer from the 7th passage 147 nucleic acid-magnetic nano particle mixture is carried out wash-out, collect from the 8th passage 148 and flow out the elutriant that contains purification of nucleic acid.
Illustrate the extracting and purifying method flow process of nucleic acid below by embodiment.
Embodiment one, and the human cervical carcinoma draws in the sea extraction purifying of (Hela) nucleus:
A, successively each passage and the micro unit of micro-fluidic chip are soaked and rinsed with distilled water, D-Hanks balanced salt solution (the D-hanks' balanced salt solution is mainly used in washed cell and tissue, is also the basal liquid of synthetic medium);
B, cultured human cervical carcinoma Hela cell is suspended in nutrient solution from the digestion of the culturing bottle of adherent culture, pass into the cell cultures micro unit 110 of micro-fluidic chip by first channel 141, cultivated 24 hours in CO2gas incubator, until cell is covered with cell cultures micro unit 110;
C, pass into pH from first channel 141 again and for the phosphate buffered saline buffer of neutral (pH value is 7.3-7.5), culturing cell is rinsed, then pass into Digestive system and cell is taken off wall digest, until cell comes off from glass substrate;
D, will digest good cell and pass into and mix micro unit 120, the cell pyrolysis liquid that will contain simultaneously the composition such as sodium laurylsulfonate from third channel 143 passes into and mixes micro unit 120, and solution mixes herein, and cell is cleaved, impels nucleic acid to be discharged in lysate;
E, pump into pH from the damping fluid passage and be the damping fluid that is suspended with ferroferric oxide magnetic nanoparticle of acid (pH is 6.3-6.7), make ferroferric oxide magnetic nanoparticle be fixed on nucleic acid by magnetic field and collect micro unit;
F, the cell pyrolysis liquid that contains nucleic acid that obtains in above-mentioned steps D is passed into nucleic acid collects micro unit 130, in the solution of pH acidity, the nucleic acid formation nucleic acid-magnetic nano particle mixture that combines with ferroferric oxide magnetic nanoparticle;
G, pass into washings from the 6th passage 146 and nucleic acid is collected the cell impurity components such as the albumen that is not attached to micro unit 130 on nano particle, lipid and washed;
H, pass into pH from the 7th passage 147 and for the damping fluid of alkalescence (pH is 8.4-8.6), nucleic acid-magnetic nano particle mixture is carried out wash-out, and collect the elutriant contain purification of nucleic acid;
I, remove magnetic field, with distilled water, whole micro-fluidic chip is washed.
Focus on a micro-fluidic chip by the collection purifying with the cultivation of cell, lysis, nucleic acid, optimized nucleic acid extraction purifying flow process, improved greatly the efficient of extracting purifying, speed and the quality of life science process have been promoted, simplify the manual operation program, avoid the use of toxic reagent, and realized the time-space resolution information that traditional method is difficult to realize, obtained nucleic acid extraction purification effect preferably.
Ferroferric oxide magnetic nanoparticle and preparation method thereof:
As shown in Figure 2, be the structural representation of ferroferric oxide magnetic nanoparticle, this nano particle comprises ferriferrous oxide nano kernel (Fe
3O
4), silicon-dioxide network molecule, amino chain hydrocarbon compound and carboxyl chain hydrocarbon compound, and amino chain hydrocarbon compound and carboxyl chain hydrocarbon compound have end of the chain amino and carboxyl, the silicon-dioxide network molecule forms shell, simultaneously crosslinked on it have amino chain hydrocarbon compound and a carboxyl chain hydrocarbon compound, ferriferrous oxide nano kernel particle diameter is 8-12nm, is wrapped to form core by the silicon-dioxide network molecule.
The preparation flow of ferroferric oxide magnetic nanoparticle comprises the steps:
The prefabricated silication ferroferric oxide nano granules inner nuclear material of 2.0ml that adds 7.0g/l in the 200ml dehydrated alcohol, and then add the 7.2ml strong aqua, add at last tetraethoxy, diethylenetriamine base propyl trimethoxy silicane and N-(the propyl trimethoxy silicane)-quadrol of different proportionings-nitrilotriacetic sodium 120ml.With the mentioned solution mixing, stopped reaction behind reaction 12h left and right under the condition that stirs, transparent state becomes translucent solution by just beginning fully, namely obtain the Z 250 silicon shell magnetic composite nano particle that the surface is modified with amino and carboxyl simultaneously, thereafter with magnetic separator wash, collecting granules is standby.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.Should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (5)
1. micro-fluidic chip, it is characterized in that, comprise the cell cultures micro unit, mix micro unit, nucleic acid is collected micro unit and microchannel, described cell cultures micro unit is used for cultivating propagation and peptic cell, to digest good cell passes into to described mixing micro unit again, described mixing micro unit is used for cracking and digests good cell, and cell pyrolysis liquid is passed into to described nucleic acid collection micro unit, described nucleic acid is collected micro unit and is used for collecting purification of nucleic acid, described microchannel connects described cell cultures micro unit, mix micro unit and nucleic acid and collect micro unit, and be the passage that liquid passes into and flows out, described micro-fluidic chip is 3 tunic structures, described 3 tunic structures comprise two-layer PDMS membrane and one deck glass substrate, and described two-layer PDMS membrane is positioned at above described glass substrate, described micro-fluidic chip also comprises air chamber, gas passage, horizontal little valve and vertical little valve, described air chamber is used for storage gas, the flow direction that gas passage is used for connecting air chamber and controls gas, described horizontal little valve and vertical little valve are controlled the flow direction of liquid in described microchannel, described air chamber, gas passage, horizontal little valve and vertical little valve are positioned between described two-layer PDMS membrane, described cell cultures micro unit, mix micro unit, nucleic acid and collect micro unit and microchannel between the space of the PDMS membrane of centre and glass substrate formation, described nucleic acid is collected micro unit and is also comprised magnetic field device, and described magnetic field device is used for the magnetic absorption ferroferric oxide magnetic nanoparticle, described ferroferric oxide magnetic nanoparticle comprises ferriferrous oxide nano kernel, silicon-dioxide network molecule, amino chain hydrocarbon compound and carboxyl chain hydrocarbon compound, and described amino chain hydrocarbon compound and described carboxyl chain hydrocarbon compound have, and the end of the chain is amino and carboxyl, silicon-dioxide network molecule form shell, crosslinked described amino chain hydrocarbon compound of while and described carboxyl chain hydrocarbon compound on it, described ferriferrous oxide nano kernel particle diameter is 8-12nm, is wrapped to form core by the silicon-dioxide network molecule.
2. utilize the nucleic acid extraction purification process of the described micro-fluidic chip of claim 1, comprise the steps:
A, cultivation contain the cell of purpose nucleic acid;
B, pass into Digestive system cell is taken off wall digestion;
C, pass into lysate postdigestive cell is carried out cracking, obtain containing the cell pyrolysis liquid of nucleic acid substances;
D, extract purification of nucleic acid from cell pyrolysis liquid;
Described step D comprises:
D1, pass into the damping fluid that is suspended with ferroferric oxide magnetic nanoparticle of pH6.3-6.7, by the fixing ferroferric oxide magnetic nanoparticle in magnetic field;
D2, will contain nucleic acid cell pyrolysis liquid by ferroferric oxide magnetic nanoparticle, under acidic conditions, nucleic acid combines formation nucleic acid-magnetic nano particle mixture and is fixed with ferroferric oxide magnetic nanoparticle;
D3, pass into washings protein, the lipid cell impurity component that is not attached on ferroferric oxide magnetic nanoparticle washed;
D4, the damping fluid that passes into pH8.4-8.6 carry out wash-out to nucleic acid-magnetic nano particle mixture, and under alkaline condition, nucleic acid separates with ferroferric oxide magnetic nanoparticle, collect the elutriant that contains purification of nucleic acid.
3. nucleic acid extraction purification process as claimed in claim 2, it is characterized in that, also comprise carry out using successively distilled water, D-Hanks balanced salt solution that micro-fluidic chip is soaked before steps A and rinse step and ending step D after remove magnetic field, use distilled water to carry out rinse step to micro-fluidic chip.
4. nucleic acid extraction purification process as claimed in claim 2, is characterized in that, passes into Digestive system in described step B and cell is taken off wall digestion comprise that also the phosphate buffered saline buffer that passes into before this neutral pH carries out rinse step to cell.
5. nucleic acid extraction purification process as described in any one in claim 2 to 4, is characterized in that, described nucleic acid is DNA.
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| CN103182333B (en) * | 2013-03-01 | 2015-07-29 | 重庆大学 | A kind of liposomal preparation and gathering-device and method |
| CN103484354B (en) * | 2013-07-16 | 2015-04-29 | 中国航天员科研训练中心 | Nucleic acid extraction chip capable of extracting nucleic acid of gram-positive bacteria and gram-negative bacteria |
| CN103667054B (en) * | 2013-09-18 | 2015-09-30 | 中国航天员科研训练中心 | A kind of integrated micro-flow control cell cultivation chip and preparation method thereof |
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| CN104560636A (en) * | 2014-12-30 | 2015-04-29 | 北京理工大学 | Cell pretreatment device and method |
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| SG10201505563XA (en) * | 2015-07-15 | 2017-02-27 | Delta Electronics Int’L Singapore Pte Ltd | Nucleic acid extraction method |
| CN108373962B (en) * | 2018-02-02 | 2021-07-09 | 太原理工大学 | Dual capture type nucleic acid extraction micro-fluidic chip |
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| JP7421469B2 (en) * | 2019-01-25 | 2024-01-24 | 積水化学工業株式会社 | Nucleic acid isolation method, nucleic acid isolation kit, and test chip |
| CN111304077B (en) * | 2020-03-03 | 2023-10-24 | 广东工业大学 | Integrated microfluidic chip for extracting nucleic acid and extraction method thereof |
| CN112481080B (en) * | 2020-12-10 | 2023-03-24 | 深圳先进技术研究院 | Micro-fluidic chip, micro-fluidic chip preparation method and nucleic acid extraction method |
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