CN110184184B - Preparation method and application of nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification - Google Patents
Preparation method and application of nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification Download PDFInfo
- Publication number
- CN110184184B CN110184184B CN201910374906.2A CN201910374906A CN110184184B CN 110184184 B CN110184184 B CN 110184184B CN 201910374906 A CN201910374906 A CN 201910374906A CN 110184184 B CN110184184 B CN 110184184B
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- reaction
- pool
- sample
- enrichment
- 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.)
- Active
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/087—Multiple sequential chambers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/12—Specific details about materials
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Clinical Laboratory Science (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Dispersion Chemistry (AREA)
- Hematology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method and application of a nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification, which is characterized by comprising the following steps: (1) designing a 3D (three-dimensional) drawing of a disc chip with 18-32 channels, and machining a disc chip die by using a machine tool; (2) heating any one of polycarbonate, polystyrene, polypropylene, polymethyl methacrylate, cycloolefin copolymer and bicycloheptene to form a colloidal liquid; (3) and (2) mixing polydimethylsiloxane, colloidal liquid and graphene oxide solution according to a volume ratio of 3-4: 1-2: 4-6, removing bubbles, pouring into a disc chip die, and removing the die after molding to obtain a transparent disc chip; (4) adding an enzyme solution containing lysozyme and proteinase k and a glutaraldehyde solution into a sample cell, and reacting at room temperature for 3-5 hours to obtain the nucleic acid reactor.
Description
Technical Field
The invention relates to a bioreactor, in particular to a preparation method and application of a three-in-one nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification.
Background
Polydimethylsiloxane (PDMS) is commonly called silicone oil, is a viscous liquid, is an organic siloxane mixture with chain structures with different polymerization degrees, has terminal groups and side groups which are all hydrocarbon groups (such as methyl, ethyl, phenyl and the like), is colorless, tasteless, nontoxic and nonvolatile, and has the working temperature of-45 to 200 ℃.
Graphene as a two-dimensional nano material has excellent optical, electrical and mechanical properties, has attracted great interest of scholars, and has become one of the research hotspots in the world. The coordination number of carbon atoms in graphene is 3, and the bond length between every two adjacent carbon atoms is 1.42 × 10-10Rice, the included angle between the keys is 120 degrees. In addition to the honeycomb layered structure in which sigma bonds are connected to other carbon atoms in a hexagonal ring, pz orbitals perpendicular to the layer plane of each carbon atom can form large pi bonds (similar to benzene rings) of multiple atoms throughout the layer, thus having excellent electrical conductive and optical properties. Graphene has chemical properties similar to graphite, and can adsorb and desorb various atoms and molecules. When the atoms or molecules are used as donors or acceptors, the concentration of graphene carriers can be changed, and graphene can keep good conductivity. But when adsorbing other species, e.g. H+And OH-In the case of graphene, some derivatives are generated, which deteriorate the conductivity of graphene, but no new compounds are generated. Therefore, the properties of graphene can be estimated using graphite. Due to the unique two-dimensional structure and excellent crystallographic quality, the graphene contains abundant and novel physical phenomena, and has important theoretical research value. The graphene oxide is obtained by oxidizing graphite with a strong oxidant such as potassium permanganate and the like and ultrasonically stripping, and the basic structure of the graphene oxide is a carbon monoatomic layer with carboxyl, hydroxyl, carbonyl and epoxy groups on the surface, contains rich oxygen-containing functional groups, is well dispersed in water and is easy to compound with other water-soluble materials. Because graphene oxide contains rich functional groups, the graphene oxide derivative can be prepared by a chemical modification method and then mixed with enzyme and eggsThe covalent bonds of the white biomacromolecules and the like are combined to construct various biochemical microreactors, and the advantages of unique property are achieved. At present, the extraction, enrichment and amplification of nucleic acid are carried out in 3 different devices, the time is long, and the operation is relatively complex.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of a nucleic acid reactor which integrates nucleic acid extraction, enrichment and in-situ amplification and can finish the processes of nucleic acid extraction, enrichment and amplification in one step and multiply the concentration index of nucleic acid in a short time and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of a nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification comprises the following steps:
(1) designing a mold: designing a disc chip 3D diagram of 18-32 channels, processing a disc chip die by a machine tool, wherein 18-32 reaction areas are radially arranged in the disc chip along the circumference, each reaction area is sequentially provided with a sample pool for breaking the wall of pathogenic microorganisms and extracting nucleic acid, a first reaction pool for enriching the nucleic acid and a second reaction pool for amplifying the nucleic acid in situ from inside to outside, sample adding holes are arranged in the sample pool, each sample adding hole of the reaction area is respectively communicated with a sample liquid storage tank at the center of the disc chip, the sample pool is communicated with the first reaction pool and the second reaction pool through micro-tube channels, the first reaction pool is communicated with a waste liquid hole, the second reaction pool is communicated with an exhaust hole for reducing the pressure of the micro-tube channels, a membrane for preventing liquid from flowing backwards is arranged on a micro-tube channel between the first reaction tank and the second reaction tank;
(2) preparation of the casting mass
a. Heating any one of Polycarbonate (PC), Polystyrene (PS), polypropylene (PP), polymethyl methacrylate (PMMA), Cyclic Olefin Copolymer (COC), and bicycloheptene (COP) to a colloidal liquid;
b. putting graphene oxide into water for ultrasonic treatment until the graphene oxide is uniformly dispersed, and then dropwise adding 5-10 drops of ammonia water to prepare a graphene oxide solution with the concentration of 0.4-1.2 g/L;
(3) casting: mixing Polydimethylsiloxane (PDMS), a colloidal liquid and a graphene oxide solution according to a volume ratio of 3-4: 1-2: 4-6, vacuumizing to remove air bubbles, pouring into a disc chip die, and removing the die after molding to obtain a transparent disc chip;
(4) adding an enzyme solution containing lysozyme and proteinase k into the sample cell of each reaction area, injecting a glutaraldehyde solution with the concentration of 0.025-0.1wt% into the sample cell of each reaction area, and reacting at room temperature for 3-5 hours to obtain the nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification.
The outer diameter of the disc chip in the step (1) is 66mm, the sample cell is oval, the short diameter is 2mm, and the long diameter is 4 mm; the diameter of the sampling hole is 1mm, and the depth of the sampling hole is 2 mm; the diameter of the first reaction tank is 2mm, and the depth of the first reaction tank is 1.5 mm; the diameter of the second reaction tank is 5mm, and the depth of the second reaction tank is 1.5 mm; the width of the micro-tube channel is 400 μm, and the depth of the micro-tube channel is 400 μm; the diameter of the waste liquid hole is 2mm, and the diameter of the exhaust hole is 1 mm; when the centrifugal force is more than 2000g, the diaphragm is opened and the first reaction tank is communicated with the second reaction tank.
The preparation method of the graphene oxide in the step (2) comprises the following steps: mixing 20 g of graphene with 100-plus-160 ml of concentrated sulfuric acid, cooling, adding 4-7 g of potassium nitrate and 20-50 g of potassium permanganate at 0-4 ℃, heating at 35-37 ℃ for 20-30 minutes to obtain viscous black brown pasty liquid, then adding 400-plus-600 ml of water, continuing heating at 95-100 ℃ for 50 minutes, adding water until the total volume of the mixed solution is 800-plus-1000 ml, adding 30-60 ml of hydrogen peroxide, performing suction filtration by using filter cloth to separate solid matters, sequentially performing suction filtration and washing by using 5wt% of dilute hydrochloric acid and water, and drying to obtain the graphene oxide.
The concentration of lysozyme in the enzyme solution in the step (4) is 100mg/mL, and the concentration of proteinase k is 20 mg/mL. Lysozyme and proteinase K are fixed by covalent bonds formed by amino and carboxyl of graphene oxide on the surface of the channel, and the proteinase is fixed on the inner wall of the sample cell by glutaraldehyde (aldehyde groups in the glutaraldehyde react with primary amine groups in the proteinase, imine groups on heterocyclic rings, hydroxyl groups, sulfydryl and amide groups to form a cyclic pyridine structure, so that the proteinase K is resistant to hydrolysis).
The nucleic acid amplification reaction method of the nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification comprises the following specific steps: adding a nucleic acid amplification reaction system containing pathogenic microorganisms into a sample solution storage tank at the center of a disc chip, respectively entering each sample pool through a sample adding hole, heating the disc chip to 35-60 ℃, and reacting for 30-40min to complete the extraction of nucleic acid; centrifuging the disc chip towards the direction of the waste liquid hole for 2-5min, allowing the liquid in the sample pool to enter a first reaction pool through a micro-tube channel, and reacting for 10-20min to complete the enrichment of nucleic acid; reversely centrifuging the disc chip for 1-2min, and allowing liquid to enter a waste liquid hole; then adding 10-15 mu L of 1M Tris-HCl solution with the pH value of 8.0 into the first reaction tank, centrifuging for 2-4min towards the direction of a waste liquid hole by the centrifugal force of more than 2000g, after the reaction liquid passes through a diaphragm and enters the second reaction tank, adding specific primers of pathogenic microorganisms, DNA polymerase and reaction buffer solution into the second reaction tank, and amplifying for 50-80min at the temperature of 60-65 ℃ to finish the nucleic acid amplification reaction.
The preparation method of the Tris-HCl solution comprises the following steps: weighing 0.61g of Tris, adding 40ml of ultrapure water for dissolution, dropwise adding concentrated HCl to adjust the pH value to 8.0, and metering to 50 ml.
Compared with the prior art, the invention has the advantages that: a three-in-one reactor integrating nucleic acid extraction, enrichment and in-situ amplification is characterized in that a transparent disc chip is made of silicon oil, graphene oxide and polycarbonate, a plurality of functional areas in the reaction area of the disc chip are connected by a microtubule channel, and lysozyme on the inner wall of a sample pool breaks the wall of pathogenic microorganisms and releases DNA to realize the extraction of pathogenic template nucleic acid. The graphene oxide on the inner wall of the first reaction tank can realize the enrichment of template DNA, and the enrichment efficiency is greatly improved (after oxidation treatment, the graphite oxide still keeps the layered structure of graphite, but a plurality of oxygen is introduced on the graphene single sheet of each layerRadical functional groups, the introduction of these oxygen-based functional groups makes the single graphene structure very complex, and can be expanded to several 10 microns in lateral dimension at any time) and the discharge of waste liquid. And after the enriched template DNA enters the second reaction tank, adding a primer, DNA polymerase and a buffer solution, realizing the in-situ exponential amplification of the DNA and greatly improving the amplification efficiency. The three-in-one reactor can realize the high-efficiency extraction of templates of 18-32 samples and the exponential amplification of DNA. Because the silicone oil, the graphene oxide and the polycarbonate contain amino groups, enzyme protein and the materials are combined together under the action of glutaraldehyde, the enzyme is fixed through a covalent bond, and the self enzymolysis of the enzyme is inhibited, so that a larger amount of the enzyme can be used, and the enzymolysis efficiency is greatly improved. The reactor can greatly reduce the enzymolysis time of protein from more than 12 hours of the traditional solution enzymolysis to within 30 minutes, greatly saves the enzymolysis time, improves the working efficiency, finishes the extraction, enrichment and amplification of nucleic acid in one step, and ensures that the concentration of target nucleic acid reaches 10 within 1-2 hours8-109 copies。
Drawings
FIG. 1 is a schematic structural diagram of a disk chip according to the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Detailed description of the preferred embodiment
A preparation method of a nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification comprises the following steps:
(1) designing a mold: designing a disc chip 3D diagram with 18-32 channels, processing a disc chip die by a machine tool, as shown in figure 1, wherein 18-32 reaction areas 1 are radially arranged on the inner edge of the disc chip along the circumference, each reaction area 1 is sequentially provided with a sample pool 2 for breaking the wall of pathogenic microorganisms and extracting nucleic acid, a first reaction pool 3 for enriching nucleic acid and a second reaction pool 4 for amplifying the nucleic acid in situ from inside to outside, sample adding holes 5 are arranged in the sample pool 2, the sample adding holes 5 of each reaction area 1 are respectively communicated with a sample liquid storage tank 6 at the center of the disc chip, the sample pool 2 and the first reaction pool 3 as well as the first reaction pool 3 and the second reaction pool 4 are communicated through a micro-tube channel 7, the first reaction pool 3 is communicated with a waste liquid hole 8, the second reaction pool 4 is communicated with an exhaust hole 9 for reducing the pressure of the micro-tube channel 7, a micro-pipe channel 7 between the first reaction tank 3 and the second reaction tank 4 is provided with a diaphragm 10 for preventing liquid from flowing backwards;
(2) preparation of the casting mass
a. Heating any one of Polycarbonate (PC), Polystyrene (PS), polypropylene (PP), polymethyl methacrylate (PMMA), Cyclic Olefin Copolymer (COC), and bicycloheptene (COP) to a colloidal liquid;
b. putting graphene oxide into water for ultrasonic treatment until the graphene oxide is uniformly dispersed, and then dropwise adding 5-10 drops of ammonia water to prepare a graphene oxide solution with the concentration of 0.4-1.2 g/L;
(3) casting: mixing Polydimethylsiloxane (PDMS), a colloidal liquid and a graphene oxide solution according to a volume ratio of 3-4: 1-2: 4-6, vacuumizing to remove air bubbles, pouring into a disc chip die, and removing the die after molding to obtain a transparent disc chip;
(4) adding an enzyme solution containing lysozyme and proteinase k into the sample cell 2 of each reaction area 1, then injecting a glutaraldehyde solution with the concentration of 0.025-0.1wt% into the sample cell 2 of each reaction area 1, and reacting for 3-5 hours at room temperature to obtain the nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification.
In this embodiment, the outer diameter of the disk chip in step (1) is 66mm, the sample cell 2 is elliptical, the short diameter is 2mm, and the long diameter is 4 mm; the diameter of the sampling hole 5 is 1mm, and the depth is 2 mm; the diameter of the first reaction tank 3 is 2mm, and the depth is 1.5 mm; the diameter of the second reaction tank 4 is 5mm, and the depth is 1.5 mm; the width of the micro-tube channel 7 is 400 μm, and the depth is 400 μm; the diameter of the waste liquid hole 8 is 2mm, and the diameter of the exhaust hole 9 is 1 mm; at a centrifugal force > 2000g, the membrane 10 is opened and the first reaction cell 3 and the second reaction cell 4 are in communication.
In this specific embodiment, the preparation method of the graphene oxide in the step (2) is as follows: mixing 20 g of graphene with 100-plus-160 ml of concentrated sulfuric acid, cooling, adding 4-7 g of potassium nitrate and 20-50 g of potassium permanganate at 0-4 ℃, heating at 35-37 ℃ for 20-30 minutes to obtain viscous black brown pasty liquid, then adding 400-plus-600 ml of water, continuing heating at 95-100 ℃ for 50 minutes, adding water until the total volume of the mixed solution is 800-plus-1000 ml, adding 30-60 ml of hydrogen peroxide, performing suction filtration by using filter cloth to separate solid matters, sequentially performing suction filtration and washing by using 5wt% of dilute hydrochloric acid and water, and drying to obtain the graphene oxide. In the step (4), the concentration of the lysozyme in the enzyme solution is 100mg/mL, and the concentration of the proteinase k is 20 mg/mL.
Detailed description of the invention
The method for nucleic acid amplification reaction of the nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification in the first embodiment comprises the following specific steps: adding a nucleic acid amplification reaction system containing pathogenic microorganisms into a sample solution storage tank 6 at the center of the disc chip, respectively entering each sample pool 2 through a sample adding hole 5, heating the disc chip to 35-60 ℃, and reacting for 30-40min to complete the extraction of nucleic acid; centrifuging the disc chip towards the direction of the waste liquid hole 8 for 2-5min, allowing the liquid in the sample pool 2 to enter the first reaction pool 3 through the micro-tube channel 7, and reacting for 10-20min to complete the enrichment of nucleic acid; reversely centrifuging the disc chip for 1-2min, and allowing liquid to enter a waste liquid hole 8; then adding 10-15 μ L of 1M Tris-HCl solution with pH of 8.0 into the first reaction tank 3, centrifuging the solution towards the direction of the waste liquid hole 8 for 2-4min by a centrifugal force larger than 2000g, after the reaction solution passes through the diaphragm 10 and enters the second reaction tank 4, adding specific primers of pathogenic microorganisms, DNA polymerase and reaction buffer solution into the second reaction tank 4, and amplifying the mixture at 60-65 ℃ for 50-80min to complete the nucleic acid amplification reaction. The preparation method of the Tris-HCl solution comprises the following steps: weighing 0.61g of Tris, adding 40ml of ultrapure water for dissolution, dropwise adding concentrated HCl to adjust the pH value to 8.0, and metering to 50 ml.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (5)
1. A preparation method of a nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification is characterized by comprising the following steps:
(1) designing a mold: designing a disc chip 3D diagram of 18-32 channels, processing a disc chip die by a machine tool, wherein 18-32 reaction areas are radially arranged in the disc chip along the circumference, each reaction area is sequentially provided with a sample pool for breaking the wall of pathogenic microorganisms and extracting nucleic acid, a first reaction pool for enriching the nucleic acid and a second reaction pool for amplifying the nucleic acid in situ from inside to outside, sample adding holes are arranged in the sample pool, each sample adding hole of the reaction area is respectively communicated with a sample liquid storage tank at the center of the disc chip, the sample pool is communicated with the first reaction pool and the second reaction pool through micro-tube channels, the first reaction pool is communicated with a waste liquid hole, the second reaction pool is communicated with an exhaust hole for reducing the pressure of the micro-tube channels, a membrane for preventing liquid from flowing backwards is arranged on a micro-tube channel between the first reaction tank and the second reaction tank;
(2) preparation of the casting mass
a. Heating any one of polycarbonate, polystyrene, polypropylene, polymethyl methacrylate, cycloolefin copolymer and bicycloheptene to form a colloidal liquid;
b. putting graphene oxide into water for ultrasonic treatment until the graphene oxide is uniformly dispersed, and then dropwise adding 5-10 drops of ammonia water to prepare a graphene oxide solution with the concentration of 0.4-1.2 g/L;
(3) casting: and (2) mixing polydimethylsiloxane, colloidal liquid and graphene oxide solution according to a volume ratio of 3-4: 1-2: 4-6, vacuumizing to remove air bubbles, pouring into a disc chip die, and removing the die after molding to obtain a transparent disc chip;
(4) adding an enzyme solution containing lysozyme and proteinase k into a sample pool of each reaction area, then injecting a glutaraldehyde solution with the concentration of 0.025-0.1wt% into the sample pool of each reaction area, and reacting for 3-5 hours at room temperature to obtain the nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification, wherein the concentration of the lysozyme in the enzyme solution is 100mg/mL, and the concentration of the proteinase k is 20 mg/mL.
2. The method for preparing a nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification as claimed in claim 1, wherein: the outer diameter of the disc chip in the step (1) is 66mm, the sample cell is oval, the short diameter is 2mm, and the long diameter is 4 mm; the diameter of the sampling hole is 1mm, and the depth of the sampling hole is 2 mm; the diameter of the first reaction tank is 2mm, and the depth of the first reaction tank is 1.5 mm; the diameter of the second reaction tank is 5mm, and the depth of the second reaction tank is 1.5 mm; the width of the micro-tube channel is 400 μm, and the depth of the micro-tube channel is 400 μm; the diameter of the waste liquid hole is 2mm, and the diameter of the exhaust hole is 1 mm; when the centrifugal force is more than 2000g, the diaphragm is opened and the first reaction tank is communicated with the second reaction tank.
3. The method for preparing a nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification as claimed in claim 1, wherein the graphene oxide in step (2) is prepared by the following steps: mixing 20 g of graphene with 100-plus-160 ml of concentrated sulfuric acid, cooling, adding 4-7 g of potassium nitrate and 20-50 g of potassium permanganate at 0-4 ℃, heating at 35-37 ℃ for 20-30 minutes to obtain viscous black brown pasty liquid, then adding 400-plus-600 ml of water, continuing heating at 95-100 ℃ for 50 minutes, adding water until the total volume of the mixed solution is 800-plus-1000 ml, adding 30-60 ml of hydrogen peroxide, performing suction filtration by using filter cloth to separate solid matters, sequentially performing suction filtration and washing by using 5wt% of dilute hydrochloric acid and water, and drying to obtain the graphene oxide.
4. The application of the nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification as claimed in any one of claims 2-3 is characterized in that the specific steps of the nucleic acid amplification reaction method are as follows: adding a nucleic acid amplification reaction system containing pathogenic microorganisms into a sample solution storage tank at the center of a disc chip, respectively entering each sample pool through a sample adding hole, heating the disc chip to 35-60 ℃, and reacting for 30-40min to complete the extraction of nucleic acid; centrifuging the disc chip towards the direction of the waste liquid hole for 2-5min, allowing the liquid in the sample pool to enter a first reaction pool through a micro-tube channel, and reacting for 10-20min to complete the enrichment of nucleic acid; reversely centrifuging the disc chip for 1-2min, and allowing liquid to enter a waste liquid hole; then adding 10-15 mu L of 1M Tris-HCl solution with the pH value of 8.0 into the first reaction tank, centrifuging for 2-4min towards the direction of a waste liquid hole by the centrifugal force of more than 2000g, after the reaction liquid passes through a diaphragm and enters the second reaction tank, adding specific primers of pathogenic microorganisms, DNA polymerase and reaction buffer solution into the second reaction tank, and amplifying for 50-80min at the temperature of 60-65 ℃ to finish the nucleic acid amplification reaction.
5. The use of a nucleic acid reactor integrating nucleic acid extraction, enrichment and in situ amplification as claimed in claim 4, wherein: the preparation method of the Tris-HCl solution comprises the following steps: weighing 0.61g of Tris, adding 40ml of ultrapure water for dissolution, dropwise adding concentrated HCl to adjust the pH value to 8.0, and metering to 50 ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374906.2A CN110184184B (en) | 2019-05-07 | 2019-05-07 | Preparation method and application of nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374906.2A CN110184184B (en) | 2019-05-07 | 2019-05-07 | Preparation method and application of nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110184184A CN110184184A (en) | 2019-08-30 |
| CN110184184B true CN110184184B (en) | 2022-03-11 |
Family
ID=67715815
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910374906.2A Active CN110184184B (en) | 2019-05-07 | 2019-05-07 | Preparation method and application of nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110184184B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808721B (en) * | 2020-07-28 | 2023-05-23 | 宁波爱基因科技有限公司 | Nucleic acid extraction chip and method for rapidly extracting blood DNA by using same |
| CN114134215A (en) * | 2021-10-29 | 2022-03-04 | 江苏鹍远生物技术有限公司 | Multichannel nucleic acid secondary amplification kit and detection method thereof |
| CN117816264B (en) * | 2024-03-01 | 2024-05-10 | 博奥生物集团有限公司 | Centrifugal microfluidic-based dual-temperature-zone type polar-speed PCR system and air compression type reaction chip |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20070052958A (en) * | 2005-11-18 | 2007-05-23 | 주식회사 엘지생명과학 | Plastic chip for pcr having on-chip polymer valve |
| CN101591014A (en) * | 2009-06-30 | 2009-12-02 | 湖北大学 | A kind of method of realizing large-scale preparation of monolayer oxidized graphene |
| CN103013824A (en) * | 2012-12-21 | 2013-04-03 | 复旦大学 | Proteolysis micro-fluidic chip based on silica gel oxidized graphene composite membrane and fabrication method of proteolysis micro-fluidic chip |
| CN104611223A (en) * | 2015-01-28 | 2015-05-13 | 中国科学院半导体研究所 | Chip and method for electrochemical detection of dPCR amplification products |
| CN105316224A (en) * | 2015-12-07 | 2016-02-10 | 中国科学院苏州生物医学工程技术研究所 | Full-automatic nucleic acid extraction and PCR amplification micro-fluidic chip and application method thereof |
| CN105372435A (en) * | 2014-08-26 | 2016-03-02 | 中国科学院苏州纳米技术与纳米仿生研究所 | Biological detection system based on graphene and making and integrating methods thereof |
| CN105420364A (en) * | 2015-12-18 | 2016-03-23 | 北京雷根生物技术有限公司 | Graphene accelerated PCR (polymerase chain reaction) technology |
| CN205347420U (en) * | 2015-12-07 | 2016-06-29 | 中国科学院苏州生物医学工程技术研究所 | Full -automatic nucleic acid extraction and PCR increase micro -fluidic chip |
| CN107051601A (en) * | 2017-06-06 | 2017-08-18 | 河南理工大学 | Detection of nucleic acids micro-fluidic chip and preparation method based on graphene field effect pipe |
| CN108043478A (en) * | 2017-12-04 | 2018-05-18 | 国家纳米科学中心 | A kind of micro-fluidic chip, manual centrifugal device and nucleic acid detection method |
| CN207850978U (en) * | 2018-03-15 | 2018-09-11 | 宁波雪弗尔生物科技有限公司 | A kind of high-throughput quantification detection closed experimental system setup of nucleic acid reaction |
| CN109613287A (en) * | 2018-12-27 | 2019-04-12 | 宁波大学 | A kind of quantitative biochip reaction apparatus |
| CN109613286A (en) * | 2018-12-27 | 2019-04-12 | 宁波大学 | A kind of qualitative/quantitative bio-chip test device |
-
2019
- 2019-05-07 CN CN201910374906.2A patent/CN110184184B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20070052958A (en) * | 2005-11-18 | 2007-05-23 | 주식회사 엘지생명과학 | Plastic chip for pcr having on-chip polymer valve |
| CN101591014A (en) * | 2009-06-30 | 2009-12-02 | 湖北大学 | A kind of method of realizing large-scale preparation of monolayer oxidized graphene |
| CN103013824A (en) * | 2012-12-21 | 2013-04-03 | 复旦大学 | Proteolysis micro-fluidic chip based on silica gel oxidized graphene composite membrane and fabrication method of proteolysis micro-fluidic chip |
| CN105372435A (en) * | 2014-08-26 | 2016-03-02 | 中国科学院苏州纳米技术与纳米仿生研究所 | Biological detection system based on graphene and making and integrating methods thereof |
| CN104611223A (en) * | 2015-01-28 | 2015-05-13 | 中国科学院半导体研究所 | Chip and method for electrochemical detection of dPCR amplification products |
| CN205347420U (en) * | 2015-12-07 | 2016-06-29 | 中国科学院苏州生物医学工程技术研究所 | Full -automatic nucleic acid extraction and PCR increase micro -fluidic chip |
| CN105316224A (en) * | 2015-12-07 | 2016-02-10 | 中国科学院苏州生物医学工程技术研究所 | Full-automatic nucleic acid extraction and PCR amplification micro-fluidic chip and application method thereof |
| CN105420364A (en) * | 2015-12-18 | 2016-03-23 | 北京雷根生物技术有限公司 | Graphene accelerated PCR (polymerase chain reaction) technology |
| CN107051601A (en) * | 2017-06-06 | 2017-08-18 | 河南理工大学 | Detection of nucleic acids micro-fluidic chip and preparation method based on graphene field effect pipe |
| CN108043478A (en) * | 2017-12-04 | 2018-05-18 | 国家纳米科学中心 | A kind of micro-fluidic chip, manual centrifugal device and nucleic acid detection method |
| CN207850978U (en) * | 2018-03-15 | 2018-09-11 | 宁波雪弗尔生物科技有限公司 | A kind of high-throughput quantification detection closed experimental system setup of nucleic acid reaction |
| CN109613287A (en) * | 2018-12-27 | 2019-04-12 | 宁波大学 | A kind of quantitative biochip reaction apparatus |
| CN109613286A (en) * | 2018-12-27 | 2019-04-12 | 宁波大学 | A kind of qualitative/quantitative bio-chip test device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110184184A (en) | 2019-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110184184B (en) | Preparation method and application of nucleic acid reactor integrating nucleic acid extraction, enrichment and in-situ amplification | |
| CN100491390C (en) | Reversible packaged micro fluent chip for separating and purifying biological sample | |
| Zhu et al. | Hydrogel droplet microfluidics for high-throughput single molecule/cell analysis | |
| Petralia et al. | PCR technologies for point of care testing: progress and perspectives | |
| CN102056838B (en) | Microfluidic device and methods | |
| Gan et al. | A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types | |
| KR101702154B1 (en) | Device for carrying out a reaction | |
| CN102465110B (en) | Cell lysis device and the method making cell or virolysis | |
| CN103451088B (en) | Micro-droplet type PCR (polymerase chain reaction) chip and manufacture method thereof | |
| CN100564541C (en) | Preprocessed chip of porous silica microfluid sample | |
| WO2015069798A1 (en) | Single-cell forensic short tandem repeat typing within microfluidic droplets | |
| AU2017202597B2 (en) | Slip chip device and methods | |
| Hwang et al. | Solid phase DNA extraction with a flexible bead-packed microfluidic device to detect methicillin-resistant Staphylococcus aureus in nasal swabs | |
| CN103221539A (en) | Protein or peptide printing method, protein or peptide array and functional protein or functional peptide identification method | |
| Tian et al. | A LAMP-based microfluidic module for rapid detection of pathogen in cryptococcal meningitis | |
| CN113025476A (en) | Double-layer micro-fluidic chip, kit and method for detecting novel coronavirus | |
| JP2004506918A (en) | Multiwell filtration plate and method of forming the same | |
| CN113026110A (en) | High-throughput single-cell transcriptome sequencing method and kit | |
| CN216274050U (en) | Multi-channel nucleic acid secondary amplification kit | |
| Choi et al. | Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure | |
| CN115728363A (en) | Method for detecting nucleic acid through CRISPR-electrochemistry based on mesoporous material release | |
| CN104342494B (en) | It is prepared by the direction specific single-chain DNA that the chemical modification primer applied to pyrosequencing mediates | |
| CN107790201A (en) | A kind of method for preparing microfluidic channel on glass using simple heat pressing process | |
| Huang et al. | An Integrated, Multiplex Digital PCR-Based Microfluidic System for Quantification of Two Microrna Biomarkers for Diagnosis of Ovarian Cancer | |
| CN113368708A (en) | Preparation method and application of suction filtration type double-layer molecular imprinting nano composite membrane based on multiple nano composite imprinting system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210128 Address after: 315040 Ningbo New Materials Innovation Center East District, Ningbo High-tech Zone, Zhejiang Province, No. 1 11-2-1 Applicant after: Zhejiang Zhenghegu Biotechnology Co.,Ltd. Address before: 315211, Fenghua Road, Jiangbei District, Zhejiang, Ningbo 818 Applicant before: Ningbo University |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |