CN114669339A - Nucleic acid extraction micro-fluidic chip, nucleic acid extraction system and method - Google Patents
Nucleic acid extraction micro-fluidic chip, nucleic acid extraction system and method Download PDFInfo
- Publication number
- CN114669339A CN114669339A CN202210584009.6A CN202210584009A CN114669339A CN 114669339 A CN114669339 A CN 114669339A CN 202210584009 A CN202210584009 A CN 202210584009A CN 114669339 A CN114669339 A CN 114669339A
- Authority
- CN
- China
- Prior art keywords
- channel
- nucleic acid
- liquid
- communicated
- acid extraction
- 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
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
-
- 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
- B01L3/50273—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 means or forces applied to move the fluids
-
- 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
- B01L3/502738—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 integrated valves
-
- 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
- B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0621—Control of the sequence of chambers filled or emptied
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/14—Process control and prevention of errors
-
- 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
- B01L2300/04—Closures and closing means
- B01L2300/046—Function or devices integrated in the closure
- B01L2300/049—Valves integrated in closure
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Dispersion Chemistry (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
A nucleic acid extraction micro-fluidic chip, a nucleic acid extraction system and a method are provided, wherein the nucleic acid extraction micro-fluidic chip comprises a top layer, a middle layer and a bottom layer, and the middle layer is provided with a first channel, a second channel and a first channel which are longitudinally arranged and mutually isolated; the bottom of the top layer is recessed at a position corresponding to the second channel to form a first recessed part, and the first recessed part is communicated with the first channel and the second channel; the top of the bottom layer is recessed at a position corresponding to the position between the second channel and the first channel to form a first groove, and the first groove is communicated with the second channel and the first channel; the middle layer is clamped between the top layer and the bottom layer, so that the first channel, the lower surface of the top layer and the upper surface of the bottom layer form a sample cavity in an enclosing manner, and the second channel, the lower surface of the top layer and the upper surface of the bottom layer form an adsorption cavity in an enclosing manner; the top layer is also provided with a first through hole communicated with the first channel as a liquid inlet, and a second through hole and a third through hole communicated with the first channel as a waste liquid port and a liquid outlet respectively. The invention has the advantages of no contact of operators with organic solvents and short extraction time.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a nucleic acid extraction micro-fluidic chip, a nucleic acid extraction system and a nucleic acid extraction method.
Background
Nucleic acid extraction is a commonly used technique in molecular biology, which is a process for obtaining high-purity nucleic acid from a complex sample. The general procedure for nucleic acid extraction comprises: 1. cell lysis, i.e., the release of nucleic acids from cells or other organisms (e.g., viruses); 2. separating and purifying nucleic acid. Methods for nucleic acid isolation and purification commonly used include: extraction and precipitation method, column extraction and magnetic bead extraction. The extraction and precipitation method refers to repeated extraction with organic solvent (such as phenol chloroform, Tizol), centrifugation, and separation, wherein nucleic acid is in the upper aqueous phase, most of denatured protein is in the middle layer, and the lower organic phase contains impurities such as lipid. Carefully absorbing the water phase, adding ethanol to precipitate the nucleic acid, washing with 70% ethanol to obtain relatively pure nucleic acid, and adding nuclease-free water to dissolve to obtain the nucleic acid. The column extraction method utilizes the characteristics that a silica gel membrane selectively adsorbs nucleic acid at high salt and low pH and releases nucleic acid at low salt and high pH to separate nucleic acid, and rinsing liquid containing ethanol is required for impurity cleaning. The magnetic bead method is to use magnetic beads as solid phase carriers to adsorb nucleic acid, usually isopropanol or polyethylene glycol is used in the binding process, and high proportion of ethanol (usually 70% -80%) is needed to wash impurities of the magnetic beads.
The existing techniques for nucleic acid extraction, regardless of the above-mentioned methods, require the use of organic solvents, such as phenol, chloroform, isopropanol, ethanol, polyethylene glycol, and the like. However, most organic solvents are toxic or have limited transportation and storage, which poses risks and inconvenience to the daily laboratory personnel. Furthermore, the nucleic acid washing requires time to evaporate after using the organic solvent so as not to affect the nucleic acid purity, and the time waiting for the evaporation usually requires 5 minutes, which is not long in appearance, but also wastes time when a large amount of samples need to be processed (such as a large amount of samples need to be extracted every day in the new crown detection).
Therefore, it is highly desirable to develop a novel method for extracting nucleic acid with good sealing properties without manual operation, so as to avoid the use of organic solvents, shorten the extraction time, and reduce the cost.
Disclosure of Invention
The invention provides a nucleic acid extraction micro-fluidic chip, a nucleic acid extraction system and a nucleic acid extraction method, which have the advantages of good tightness and short extraction time.
In order to achieve the above purpose, the invention provides a nucleic acid extraction microfluidic chip, which comprises a top layer, a middle layer and a bottom layer, wherein the middle layer is provided with a first channel, a second channel and a first channel which are longitudinally arranged and mutually isolated; the bottom of the top layer is recessed at a position corresponding to the second channel to form a first recessed part, and the first recessed part is communicated with the first channel and the second channel; the top of the bottom layer is recessed at a position corresponding to the position between the second channel and the first channel to form a first groove, and the first groove is communicated with the second channel and the first channel; the middle layer is clamped between the top layer and the bottom layer, so that the first channel, the lower surface of the top layer and the upper surface of the bottom layer form a sample cavity in an enclosing manner, and the second channel, the lower surface of the top layer and the upper surface of the bottom layer form an adsorption cavity in an enclosing manner; the top layer is also provided with a first through hole communicated with the first channel as a liquid inlet, and a second through hole and a third through hole communicated with the first channel as a waste liquid port and a liquid outlet respectively.
Furthermore, the front part of the first channel is in a shape with gradually increased width, and the first through hole is communicated with the foremost end of the first channel, so that the liquid entering from the first through hole moves in a fan shape in the first channel.
Furthermore, the rear part of the first channel comprises a second channel with the width smaller than that of the middle part of the first channel, and a transition section with the shape of gradually decreasing width is arranged between the middle part of the first channel and the second channel.
Furthermore, the front part of the second channel is in an increasing width shape, the rear part of the second channel is in a decreasing width shape, the first concave part comprises a concave part main body which is the same as the second channel in shape and a connecting groove part which is positioned at the front part of the concave part main body and is in a channel shape, and the first concave part is communicated with the first channel through the connecting groove part.
Further, the first channel comprises a longitudinal channel part and a transverse channel part which are communicated, the longitudinal channel part is communicated with the waste liquid port, and the transverse channel part is communicated with the liquid outlet.
Furthermore, the middle layer is of a frame structure and is fixed with the bottom layer through a positioning hole.
Furthermore, the middle layer is also provided with a third channel and a fourth channel which are connected with the first channel at the rear part of the first channel, and the top layer is also provided with a fourth through hole and a fifth through hole which are communicated with the third channel and the fourth channel and are used as liquid inlets.
The invention also provides a nucleic acid extraction system, which is used for extracting a reagent from a lysis mixture container, a cleaning solution container and an eluent container respectively, extracting an extracting solution after the reagent is injected, and removing redundant liquid into the extracting solution container and a waste liquid container, wherein the nucleic acid extraction system comprises the nucleic acid extraction micro-fluidic chip, an injection pump, a shear valve and a distribution valve, the injection pump is connected with the distribution valve, each valve port of the distribution valve is respectively connected with a nucleic acid extraction micro-fluidic chip waste liquid port, the extracting solution container and the waste liquid container, each valve port of the shear valve is respectively connected with the lysis mixture container, the cleaning solution container and the eluent container and the outside, an outlet is connected with a nucleic acid extraction micro-fluidic chip liquid inlet, a nucleic acid extraction micro-fluidic chip liquid outlet is connected with the extracting solution container, and liquid is driven to flow into the nucleic acid extraction micro-fluidic chip from the lysis mixture container, the cleaning solution container or the eluent container by negative pressure of the injection pump, or the liquid flows into the injection pump from the waste liquid port of the nucleic acid extraction micro-fluidic chip, or flows into the extracting solution container from the liquid outlet of the nucleic acid extraction micro-fluidic chip, and the liquid is driven by the positive pressure of the injection pump to flow into the waste liquid container from the injection pump.
The device further comprises a control device and a processing device, wherein the control device is connected with the processing device, the injection pump, the shear valve and the distribution valve, and sends an instruction to the control device through the program setting of the processing device, and the control device further controls the actions of the injection pump, the shear valve and the distribution valve to realize the automatic control of the nucleic acid extraction.
The present invention also provides a nucleic acid extraction method using the nucleic acid extraction system as described above, the method including:
the method comprises the following steps that firstly, a shear valve is communicated with a cracking mixed liquid container, and a distribution valve is communicated with a waste liquid port of a nucleic acid extraction micro-fluidic chip; the injection pump runs for a certain stroke under negative pressure, so that a certain amount of cracking mixed liquid enters the sample cavity through the liquid adding port; the distribution valve is communicated with the waste liquid container; resetting the injection pump, and if the injection pump has redundant liquid, discharging the redundant liquid to a waste liquid container;
secondly, the shear valve is communicated with a cleaning fluid container, and the distribution valve is communicated with a waste fluid port of the nucleic acid extraction microfluidic chip; the injection pump runs for a certain stroke under negative pressure, so that the cracking mixed liquid is moved into the adsorption cavity from the sample cavity; the distribution valve is communicated with the waste liquid container; resetting the injection pump, and if the injection pump has redundant liquid, discharging the redundant liquid to a waste liquid container;
thirdly, the shear valve is communicated with a cleaning liquid container, and the distribution valve is communicated with a waste liquid port of the nucleic acid extraction microfluidic chip; the injection pump runs for a certain stroke under negative pressure, so that a certain amount of cleaning liquid enters the adsorption cavity through the liquid adding port; the distribution valve is communicated with the waste liquid container; resetting the injection pump, and discharging the redundant liquid to a waste liquid container;
step four, the shear valve is communicated with the outside, and the distribution valve is communicated with a waste liquid port of the nucleic acid extraction microfluidic chip; the injection pump runs for a certain stroke under negative pressure, so that air enters the sample cavity from the liquid adding port and is discharged to the injection pump from the waste liquid port; the distribution valve is communicated with the waste liquid container; resetting the injection pump;
step five, the shear valve is communicated with an eluent container, and the distribution valve is communicated with an extracting solution container; the injection pump runs for a certain stroke under negative pressure, so that a certain amount of elution liquid enters the adsorption cavity through the liquid adding port, and a certain amount of extracting solution flows out of the liquid outlet to the extracting solution container; the distribution valve is communicated with the waste liquid container; the syringe pump is reset and excess liquid is discharged to the waste container.
The invention has the beneficial effects that: according to the micro-fluidic chip, the nucleic acid extraction system and the method, the top layer, the middle layer and the bottom layer which are mutually overlapped and the special structure thereof are arranged to form the sample cavity, the adsorption cavity, the liquid inlet, the waste liquid port and the liquid outlet, so that the cracking mixed liquid is cleaned, eluted and the like in a closed space, and an operator does not need to contact with an organic solvent; the structure is compact, the operation is convenient, and the extraction time can be obviously shortened; simple structure, few parts and low cost.
Drawings
FIG. 1 is an exploded view of a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention.
FIG. 2 is an exploded view of the nucleic acid extraction microfluidic chip shown in FIG. 1.
FIG. 3 is an exploded view of the middle layer and the bottom layer of the nucleic acid extraction microfluidic chip shown in FIG. 1.
FIG. 4 is a cross-sectional view of the nucleic acid extraction microfluidic chip shown in FIG. 1.
FIG. 5 is a schematic diagram of a nucleic acid extraction system according to an embodiment of the present invention.
FIG. 6 is a schematic flow chart of a nucleic acid extraction method according to an embodiment of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings and examples.
As shown in fig. 1 to 4, in the present embodiment, the nucleic acid extraction microfluidic chip includes a top layer 1, an intermediate layer 2, and a bottom layer 3. The top layer 1, the middle layer 2 and the bottom layer 3 are mutually superposed, and the middle layer 2 is clamped between the top layer 1 and the bottom layer 3. The top layer, the middle layer and the bottom layer are preferably made of PMMA materials with a hydrophilic angle of 60 degrees.
In this embodiment, the middle layer 2 is a frame structure, and each end thereof is provided with a positioning hole 21 to be fixed with the bottom layer 3. The middle layer 2 is provided with a first channel 22, a second channel 23 and a first channel 24 which are arranged longitudinally and are mutually separated. The first passage 22 and the second passage 23, and the second passage 23 and the first groove 24 are isolated by a first transverse isolation part 25 and a second transverse isolation part 26 respectively, so that the first passage 22, the second passage 23 and the first groove 24 are not communicated with each other.
The first channel 22 is substantially in a flat hexagon shape, the front part is in a shape of increasing width, the middle part is in a rectangular shape, the rear part comprises a second channel 221 with the width smaller than that of the middle part of the first channel, and a transition section in a shape of decreasing width is arranged between the middle part of the first channel and the second channel 221. The front part, the middle part and the transition section form a hexagon.
The second channel 23 is substantially hexagonal, with a front portion of increasing width, a rear portion of decreasing width and a central portion of substantially square shape.
The first channel 24 includes a longitudinal channel portion 241 and a transverse channel portion 242 in communication.
The top layer 1 is a plate-shaped structure, the bottom of the top layer is recessed at a position corresponding to the second channel 23 to form a first recessed portion 11, and the first recessed portion 11 comprises a connecting groove portion 111 at the front end and a recessed portion main body 112 at the rear end. The main body of the depressed part and the second channel are basically the same in shape and size, and the connecting groove part is arranged at a corresponding position between the first channel and the second channel (namely, above the first transverse partition part). The top layer 1 is further provided with a first through hole 12, a second through hole 13 and a third through hole 14 which are vertically penetrated, and the positions of the first through hole, the second through hole and the third through hole correspond to the foremost end of the first passage 22, the tail end of the longitudinal channel part 241 and the tail end of the transverse channel part 242 respectively. The first through hole 12, the second through hole 13 and the third through hole 14 can be inserted with the butt joint 4 to facilitate the insertion of the connecting pipe.
The bottom layer 3 is a plate-like structure, the top of which is recessed to form a first recess 31 in a position corresponding to between the second channel 23 and the first channel 24 (i.e. below the second lateral partition). The bottom layer 3 is provided with a plurality of positioning columns 32 corresponding to the positioning holes 21, and the periphery of the positioning columns is fixedly connected with the top layer through screws.
After the nucleic acid extraction microfluidic chip is assembled, the first through hole in the top layer is communicated with the foremost end of the first channel in the middle layer to be used as a liquid inlet, and the second through hole and the third through hole are respectively used as a waste liquid port and a liquid outlet so as to discharge redundant liquid and extracting solution. The first channel, the lower surface of the top layer and the upper surface of the bottom layer form a sample cavity in a surrounding mode, and the second channel, the lower surface of the top layer and the upper surface of the bottom layer form an adsorption cavity in a surrounding mode. Because the front part of the first channel is in the shape of gradually increasing width, the liquid entering from the first through hole can diffuse around and move in a fan shape when flowing, thereby having higher flowing speed and large spreading area. The first recess and the second channel have the same shape with increasing width. The first sunken part connecting groove part at the bottom of the top layer is positioned above the transverse isolating part between the first channel and the second channel of the middle layer, the first channel and the second channel are communicated, a structure similar to a communicating device is formed, and the effect of preventing liquid from directly entering the adsorption cavity without filling the sample cavity is achieved. The first groove on the top of the bottom layer is positioned below the second transverse isolating part and is communicated with the second channel and the first channel, so that liquid can be prevented from being sucked out of the chip without filling the adsorption cavity.
In this embodiment, the middle layer 2 may further have a third channel 27 and a fourth channel 28 connecting the first channel 22 at the rear of the first channel 22, and the top layer 1 further has a fourth through hole 15 and a fifth through hole 16 communicating the third channel 27 and the fourth channel 28 as liquid inlets. The first through hole can be used as a uniform liquid inlet for various reagents (at this time, the fourth through hole, the fifth through hole, the third channel and the fourth channel are not required to be arranged), and the first through hole, the fourth through hole and the fifth through hole can also be used as liquid inlets for various reagents respectively.
As shown in fig. 5, in the present embodiment, the nucleic acid extraction system including the above-mentioned nucleic acid extraction microfluidic chip is used for extracting the extraction liquid into the extraction liquid container 54 and removing the excess liquid into the waste liquid container 55 after the injection of the reagent is extracted from the lysis mixture container 51, the cleaning liquid container 52 and the eluent container 53, respectively. The lysis mixture container contains a lysis mixture, which is a mixture of lysis solution and a sample to be extracted (e.g. blood). The cleaning solution container is filled with cleaning solution (such as ethanol) for cleaning impurities to obtain relatively pure nucleic acid. The eluent is filled in the eluent container, so that the nucleic acid adsorbed on the silica gel membrane in the adsorption cavity is released in a low-salt high-pH environment.
The nucleic acid extraction system also comprises an injection pump 71, a shear valve 6, a distribution valve 7, a control device 8 and a processing device 9, wherein the injection pump 71 is connected with the distribution valve 7, each valve port (E, I, O) of the distribution valve 7 is respectively connected with the second through hole 13 of the nucleic acid extraction microfluidic chip, the extracting solution container 54 and the waste liquid container 55, each valve port (A, B, C, D) of the shear valve 6 is respectively connected with the lysis mixture container 51, the cleaning solution container 52 and the eluent container 53 and the outside, and the outlet is connected with the first through hole 12 of the nucleic acid extraction microfluidic chip. The outlet of the switching valve 6 and the first through hole 12 can be provided with a flow meter 10 to accurately meter the amount of liquid being carried out. The third through hole 14 of the nucleic acid extraction microfluidic chip is connected with the extracting solution container 54, and liquid is driven to flow into the nucleic acid extraction microfluidic chip from the lysis mixture container, the cleaning solution container or the eluent container under negative pressure by the injection pump 71, or flow into the injection pump from a waste liquid port of the nucleic acid extraction microfluidic chip, or flow into the extracting solution container from a liquid outlet of the nucleic acid extraction microfluidic chip; liquid is positively driven from the syringe pump into the waste reservoir by the syringe pump. The adsorption cavity of the nucleic acid extraction microfluidic chip is provided with a silica gel membrane (also called as an "adsorption membrane") for adsorbing nucleic acid (in a high-salt low-pH environment).
The control unit 8 is connected to the processing unit 9, the syringe pump 71, the shear valve 6, and the distribution valve 7, and gives instructions to the control unit by program setting of the processing unit, and the control unit further controls the operations of the syringe pump, the shear valve, and the distribution valve, thereby realizing automatic control of nucleic acid extraction. The processing device can be a computer and programming software installed in the computer, programs are compiled through the programming software, and instructions are sent to the control device during running so as to realize automatic control. Of course, in other embodiments, the control device and the processing device may not be provided, and the syringe pump, the switching valve and the dispensing valve may be manually controlled by an operator.
In the embodiment, the cleavage mixture container, the cleaning solution container and the eluent container are switched and communicated by using the switching valve, so that the cleavage mixture, the cleaning solution and the eluent enter the nucleic acid extraction microfluidic chip through the first through hole, the control is concentrated, and the automatic control is more conveniently realized. In other embodiments, the fourth through hole and the fifth through hole can also be used as liquid inlets, for example, the first through hole is connected to the lysis mixture container, the fourth through hole is connected to the cleaning liquid container, and the fifth through hole is connected to the eluent container, so that the structure is simpler.
As shown in FIG. 6, in this embodiment, the method for extracting nucleic acid using the nucleic acid extraction system as described above comprises the following steps:
step one, enabling a lysis mixed solution to enter a sample cavity: the shear valve is communicated with a cracking mixed liquid container (namely, the opening A of the shear valve 6 in the figure 5 is communicated), and the distribution valve is communicated with a waste liquid opening of the nucleic acid extraction microfluidic chip (namely, the opening E of the distribution valve 7 in the figure 5 is communicated); the injection pump runs for a certain stroke under negative pressure, so that a certain amount (such as 60 uL) of cracking mixed liquid enters the sample cavity through the liquid adding port; the distribution valve is communicated with a waste liquid container (namely, the O port of the distribution valve 7 in the figure 5 is communicated); resetting the injection pump, and if the injection pump has redundant liquid, discharging the redundant liquid to a waste liquid container;
step two, moving the lysis mixture from the sample cavity into the adsorption cavity: the switching valve is communicated with a cleaning solution container (namely, the opening B of the switching valve 6 in the figure 5 is communicated), and the distribution valve is communicated with a waste solution opening of the nucleic acid extraction microfluidic chip (namely, the opening E of the distribution valve 7 in the figure 5 is communicated); the injection pump runs for a certain stroke under negative pressure, so that the cracking mixed liquid is moved into the adsorption cavity from the sample cavity; the distribution valve is communicated with a waste liquid container (namely, the O port of the distribution valve 7 in the figure 5 is communicated); resetting the injection pump, and if the injection pump has redundant liquid, discharging the redundant liquid to a waste liquid container; in the step, the cracking mixed solution is moved into the adsorption cavity from the sample cavity, and the nucleic acid is attached to the silica gel membrane;
step three, adding a cleaning solution: the switching valve is communicated with a cleaning solution container (namely, the opening B of the switching valve 6 in the figure 5 is communicated), and the distribution valve is communicated with a waste solution opening of the nucleic acid extraction microfluidic chip (namely, the opening E of the distribution valve 7 in the figure 5 is communicated); the injection pump runs for a certain stroke under negative pressure, so that a certain amount (such as 200 uL) of cleaning liquid enters the adsorption cavity through the liquid adding port; the distribution valve is communicated with a waste liquid container (namely, the O port of the distribution valve 7 in the figure 5 is communicated); resetting the injection pump, and discharging the redundant liquid to a waste liquid container; the step is used for cleaning impurities in the cracking mixed liquor;
step four, adding air: the shear valve is communicated with the outside (namely, the D port of the shear valve 6 in the figure 5 is communicated), and the distribution valve is communicated with the waste liquid port of the nucleic acid extraction microfluidic chip (namely, the E port of the distribution valve 7 in the figure 5 is communicated); the injection pump runs for a certain stroke under negative pressure, so that air enters the sample cavity from the liquid adding port and is discharged to the injection pump from the waste liquid port; the distribution valve is communicated with a waste liquid container (namely, the O port of the distribution valve 7 in the figure 5 is communicated); resetting the injection pump; this step can be repeated for 2-3 minutes; this step is used to volatilize the cleaning solution (such as ethanol);
adding eluent and extracting: the switching valve is communicated with an eluent container (namely, the opening C of the switching valve 6 in the figure 5 is communicated), and the distribution valve is communicated with an extracting solution container (namely, the opening I of the distribution valve 7 in the figure 5 is communicated); the injection pump operates at negative pressure for a certain stroke, so that a certain amount (such as 60 uL) of elution liquid enters the adsorption cavity through the liquid adding port, and a certain amount (such as 20 uL) of extracting solution flows out of the liquid outlet to the extracting solution container; the distributing valve is communicated with a waste liquid container (namely, the O port of the distributing valve 7 in the figure 5 is communicated); resetting the injection pump, and discharging the redundant liquid to a waste liquid container; the step is used for releasing and extracting the nucleic acid adsorbed on the silica gel membrane in the adsorption cavity.
The above steps can be realized by writing corresponding programs in the computer by programming software, converting the programs into instructions during running and sending the instructions to the control device, and then controlling the actions of the injection pump, the switching valve and the distribution valve by the control device so as to realize the automatic control of nucleic acid extraction.
Claims (10)
1. A nucleic acid extraction micro-fluidic chip is characterized by comprising a top layer, a middle layer and a bottom layer, wherein the middle layer is provided with a first channel, a second channel and a first channel which are longitudinally arranged and mutually isolated; the bottom of the top layer is recessed at a position corresponding to the second channel to form a first recessed part, and the first recessed part is communicated with the first channel and the second channel; the top of the bottom layer is recessed at a position corresponding to the position between the second channel and the first channel to form a first groove, and the first groove is communicated with the second channel and the first channel; the middle layer is clamped between the top layer and the bottom layer, so that the first channel, the lower surface of the top layer and the upper surface of the bottom layer form a sample cavity in an enclosing manner, and the second channel, the lower surface of the top layer and the upper surface of the bottom layer form an adsorption cavity in an enclosing manner; the top layer is also provided with a first through hole communicated with the first channel as a liquid inlet, and a second through hole and a third through hole communicated with the first channel as a waste liquid port and a liquid outlet respectively.
2. The nucleic acid extraction microfluidic chip of claim 1, wherein the front portion of the first channel is gradually increased in width, and the first through hole is communicated with the foremost end of the first channel, so that the liquid entering from the first through hole moves in a fan shape in the first channel.
3. The nucleic acid extraction microfluidic chip of claim 1, wherein the rear portion of the first channel comprises a second channel having a smaller width than the middle portion of the first channel, and a transition section having a shape with a decreasing width is disposed between the middle portion of the first channel and the second channel.
4. The nucleic acid extraction microfluidic chip of claim 1, wherein the front portion of the second channel has an increasing width, the rear portion of the second channel has a decreasing width, the first recess comprises a recess body having the same shape as the second channel and a channel-shaped connection groove portion located at the front portion of the recess body, and the first recess is in communication with the first channel through the channel-shaped connection groove portion.
5. The nucleic acid extraction microfluidic chip of claim 1, wherein the first channel comprises a longitudinal channel portion and a transverse channel portion, the longitudinal channel portion and the transverse channel portion are communicated, the longitudinal channel portion is communicated with the waste liquid port, and the transverse channel portion is communicated with the liquid outlet port.
6. The nucleic acid extraction microfluidic chip of claim 1, wherein the middle layer is a frame structure and is fixed to the bottom layer through a positioning hole.
7. The nucleic acid extraction microfluidic chip of claim 1, wherein the middle layer is further provided with a third channel and a fourth channel connecting the first channel at the rear part of the first channel, and the top layer is further provided with a fourth through hole and a fifth through hole communicating the third channel and the fourth channel as liquid inlets.
8. A nucleic acid extraction system is characterized in that the system is used for extracting a reagent from a lysis mixture container, a cleaning solution container and an eluent container respectively, extracting an extracting solution after the reagent is injected and removing redundant liquid into the extracting solution container and a waste liquid container, the nucleic acid extraction system comprises the nucleic acid extraction microfluidic chip as claimed in any one of claims 1 to 6, an injection pump, a shear valve and a distribution valve, the injection pump is connected with the distribution valve, each valve port of the distribution valve is respectively connected with a nucleic acid extraction microfluidic chip waste liquid port, the extracting solution container and the waste liquid container, each valve port of the shear valve is respectively connected with the lysis mixture container, the cleaning solution container and the eluent container and the outside, an outlet is connected with a nucleic acid extraction microfluidic chip liquid inlet, a nucleic acid extraction microfluidic chip liquid outlet is connected with the extracting solution container, and liquid is driven to flow into the nucleic acid extraction microfluidic chip from the lysis mixture container, the cleaning solution container or the eluent container by negative pressure of the injection pump, or the liquid flows into the injection pump from the waste liquid port of the nucleic acid extraction micro-fluidic chip, or flows into the extracting solution container from the liquid outlet of the nucleic acid extraction micro-fluidic chip, and the liquid is driven by the positive pressure of the injection pump to flow into the waste liquid container from the injection pump.
9. The nucleic acid isolation system according to claim 8, further comprising a control unit and a processing unit, wherein the control unit is connected to the processing unit, the syringe pump, the shear valve, and the distribution valve, and the control unit is configured to issue a command to the control unit according to a program of the processing unit, and the control unit further controls the operation of the syringe pump, the shear valve, and the distribution valve, thereby automatically controlling the nucleic acid isolation.
10. A method for nucleic acid extraction using the nucleic acid extraction system according to claim 8 or 9, the method comprising:
the method comprises the following steps that firstly, a shear valve is communicated with a cracking mixed liquid container, and a distribution valve is communicated with a waste liquid port of a nucleic acid extraction micro-fluidic chip; the injection pump runs for a certain stroke under negative pressure, so that a certain amount of cracking mixed liquid enters the sample cavity through the liquid adding port; the distribution valve is communicated with the waste liquid container; resetting the injection pump, and if the injection pump has redundant liquid, discharging the redundant liquid to a waste liquid container;
secondly, the shear valve is communicated with a cleaning fluid container, and the distribution valve is communicated with a waste fluid port of the nucleic acid extraction microfluidic chip; the injection pump runs for a certain stroke under negative pressure, so that the cracking mixed liquid is moved into the adsorption cavity from the sample cavity; the distribution valve is communicated with the waste liquid container; resetting the injection pump, and if the injection pump has redundant liquid, discharging the redundant liquid to a waste liquid container;
thirdly, the shear valve is communicated with a cleaning liquid container, and the distribution valve is communicated with a waste liquid port of the nucleic acid extraction microfluidic chip; the injection pump runs for a certain stroke under negative pressure, so that a certain amount of cleaning liquid enters the adsorption cavity through the liquid adding port; the distribution valve is communicated with the waste liquid container; resetting the injection pump, and discharging the redundant liquid to a waste liquid container;
step four, the shear valve is communicated with the outside, and the distribution valve is communicated with a waste liquid port of the nucleic acid extraction microfluidic chip; the injection pump runs for a certain stroke under negative pressure, so that air enters the sample cavity from the liquid adding port and is discharged to the injection pump from the waste liquid port; the distribution valve is communicated with the waste liquid container; resetting the injection pump;
step five, the shear valve is communicated with an eluent container, and the distribution valve is communicated with an extracting solution container; the injection pump operates at negative pressure for a certain stroke, so that a certain amount of elution liquid enters the adsorption cavity through the liquid adding port, and a certain amount of extracting solution flows out of the liquid outlet to the extracting solution container; the distribution valve is communicated with the waste liquid container; the syringe pump is reset and excess liquid is discharged to the waste container.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210584009.6A CN114669339B (en) | 2022-05-27 | 2022-05-27 | Nucleic acid extraction micro-fluidic chip, nucleic acid extraction system and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210584009.6A CN114669339B (en) | 2022-05-27 | 2022-05-27 | Nucleic acid extraction micro-fluidic chip, nucleic acid extraction system and method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114669339A true CN114669339A (en) | 2022-06-28 |
| CN114669339B CN114669339B (en) | 2022-09-09 |
Family
ID=82079352
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210584009.6A Active CN114669339B (en) | 2022-05-27 | 2022-05-27 | Nucleic acid extraction micro-fluidic chip, nucleic acid extraction system and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114669339B (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140154688A1 (en) * | 2012-12-03 | 2014-06-05 | Samsung Electronics Co. Ltd. | Reagent container for amplifying nucleic acid, method of preparing the reagent container, method of storing the reagent, and microfluidic system for nucleic acid analysis |
| CN104236787A (en) * | 2014-09-05 | 2014-12-24 | 龙微科技无锡有限公司 | Mems differential pressure sensor chip and manufacturing method |
| CN104588139A (en) * | 2015-01-20 | 2015-05-06 | 重庆科技学院 | Micro-fluidic chip for preparing microspheres and using method of micro-fluidic chip |
| CN110653015A (en) * | 2019-10-30 | 2020-01-07 | 潍坊医学院 | Microbial detection chip with high sample filling rate and filling method thereof |
| CN111607483A (en) * | 2020-05-07 | 2020-09-01 | 广州再生医学与健康广东省实验室 | In-vitro diagnostic instrument and extraction device |
| CN111774111A (en) * | 2020-07-10 | 2020-10-16 | 上海佰芯生物科技有限公司 | Micro-fluidic chip for detecting glycosylated hemoglobin and detection method thereof |
| CN112375669A (en) * | 2020-10-30 | 2021-02-19 | 清华大学深圳国际研究生院 | Micro-fluidic chip for extracting, purifying and detecting nucleic acid |
| CN112730560A (en) * | 2020-12-10 | 2021-04-30 | 东南大学 | Micro-fluidic impedance cell instrument and preparation method thereof |
| CN114182000A (en) * | 2021-12-10 | 2022-03-15 | 西安交通大学 | Integrated nucleic acid detection chip and method based on CRISPR technology |
| CN114308145A (en) * | 2020-09-30 | 2022-04-12 | 新加坡正煦诊断有限公司 | Microfluidic device, kit and preparation method thereof |
-
2022
- 2022-05-27 CN CN202210584009.6A patent/CN114669339B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140154688A1 (en) * | 2012-12-03 | 2014-06-05 | Samsung Electronics Co. Ltd. | Reagent container for amplifying nucleic acid, method of preparing the reagent container, method of storing the reagent, and microfluidic system for nucleic acid analysis |
| CN104236787A (en) * | 2014-09-05 | 2014-12-24 | 龙微科技无锡有限公司 | Mems differential pressure sensor chip and manufacturing method |
| CN104588139A (en) * | 2015-01-20 | 2015-05-06 | 重庆科技学院 | Micro-fluidic chip for preparing microspheres and using method of micro-fluidic chip |
| CN110653015A (en) * | 2019-10-30 | 2020-01-07 | 潍坊医学院 | Microbial detection chip with high sample filling rate and filling method thereof |
| CN111607483A (en) * | 2020-05-07 | 2020-09-01 | 广州再生医学与健康广东省实验室 | In-vitro diagnostic instrument and extraction device |
| CN111774111A (en) * | 2020-07-10 | 2020-10-16 | 上海佰芯生物科技有限公司 | Micro-fluidic chip for detecting glycosylated hemoglobin and detection method thereof |
| CN114308145A (en) * | 2020-09-30 | 2022-04-12 | 新加坡正煦诊断有限公司 | Microfluidic device, kit and preparation method thereof |
| CN112375669A (en) * | 2020-10-30 | 2021-02-19 | 清华大学深圳国际研究生院 | Micro-fluidic chip for extracting, purifying and detecting nucleic acid |
| CN112730560A (en) * | 2020-12-10 | 2021-04-30 | 东南大学 | Micro-fluidic impedance cell instrument and preparation method thereof |
| CN114182000A (en) * | 2021-12-10 | 2022-03-15 | 西安交通大学 | Integrated nucleic acid detection chip and method based on CRISPR technology |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114669339B (en) | 2022-09-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9506847B2 (en) | Method and system for selective isolation of target biological molecules in a general purpose system | |
| US6232464B1 (en) | Process and apparatus for isolating nucleic acids | |
| US8629264B2 (en) | Gravity flow fluidic device for nucleic acid extraction | |
| CN108893462B (en) | Device and method for preparing a biological sample, in particular for extracting DNA, and for loading the sample in a well for subsequent PCR | |
| US20060222569A1 (en) | Device and method for the preparation of analyte comprising liquids | |
| US20080153078A1 (en) | System for isolating biomolecules from a sample | |
| US20090095057A1 (en) | Integrated microfluidic nozzle device for chromatographic sample preparation for mass spectrometry applications | |
| US8017379B2 (en) | Nucleic acid purification instrument | |
| US11905550B2 (en) | Process for preparing a biological sample | |
| WO2009098104A1 (en) | Method and device for the automated processing of a sample | |
| JP2010091568A (en) | Pipette tip part having separating material | |
| CN109456880B (en) | On-site rapid nucleic acid extraction tube and use method thereof | |
| EP1203080B1 (en) | Isolation of biomolecules | |
| WO2014048263A1 (en) | Method for pressure extraction of nucleic acids and device thereof | |
| CN105733923B (en) | A kind of micro-fluidic chip and the nucleic acid extraction purification process using micro-fluidic chip | |
| US20150024384A1 (en) | Sequential delivery device and method | |
| CN113244970A (en) | Microfluidic chip for nucleic acid extraction and PCR detection and application thereof | |
| CN114669339B (en) | Nucleic acid extraction micro-fluidic chip, nucleic acid extraction system and method | |
| JP2004004078A (en) | Blocking preventing device and method for application to gel digestion | |
| CN203803213U (en) | Filter holding barrel, filter holding barrel component and rotating column component | |
| CN115029210A (en) | Closed type rapid nucleic acid extraction device and extraction method adopting one-step method | |
| CN114164102A (en) | Micro-fluidic chip for nucleic acid purification and nucleic acid purification method | |
| CN205223192U (en) | Nucleic acid extraction device | |
| JP2005320322A (en) | Fractionation apparatus | |
| CN212833710U (en) | Simple nucleic acid extraction device |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |