CN108728327A - A kind of full-automatic sample preparation microfluidic system and preparation method thereof and application method - Google Patents
A kind of full-automatic sample preparation microfluidic system and preparation method thereof and application method Download PDFInfo
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- CN108728327A CN108728327A CN201810502150.0A CN201810502150A CN108728327A CN 108728327 A CN108728327 A CN 108728327A CN 201810502150 A CN201810502150 A CN 201810502150A CN 108728327 A CN108728327 A CN 108728327A
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- storage pool
- lysate
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- amplifing reagent
- microfluidic system
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
The invention discloses a kind of full-automatic sample preparation microfluidic system and preparation method thereof and application methods, the system includes chip body and sample interface module, and concatenated entrance, the first chip valve, amplification chamber, the second chip valve, product delivery outlet and waste liquid delivery outlet are disposed in chip body；Sample interface module is tightly connected with chip body, and sample interface module includes that the upper and lower lysate storage pool arranged and sealed off and amplifing reagent storage pool, the lower end of amplifing reagent storage pool are isolated with inlet seal；Above system and have unique sample-adding module and liquid storage pattern, integrated automation processing can be carried out to biological sample, directly give amplified production, whole process is completely without with additional the laboratory special equipment or instrument such as pipettor, shaker, centrifuge, the preparation flow is allow to be detached from laboratory environment progress, it is easy to operate, it can be completed by the layman that slightly trains, make it possible the Site Detection of DNA.
Description
Technical field
The present invention relates to biochip technology field, more particularly to a kind of full-automatic sample preparation microfluidic system and its system
Preparation Method and application method.
Background technology
For DNA detection techniques, preposition sample preparation procedure is particularly important.Sample preparation generally comprises carefully
Cellular lysate, DNA extractions, purifying and DNA cloning these steps.
Existing fully integrated specimen preparation system is generally based on the high-throughput sample preparation instrument of liquid working standing posture.It is right
For liquid working station, principle is the manipulator that the reagent of human configuration, transfer will be needed to be transplanted to fixed formula originally
In there is instrument to be automatically performed, existing liquid working station mainly for DNA extract, purifying and PCR system configure these rings
It saves to run, generally without cell cracking and PCR amplification function, integrated level is not high, it is often also needed in preparation process
The addition of his special equipment such as centrifuge, whirlpool concussion instrument, pipettor, PCR thermal cyclers etc. could be completed, therefore whole operation
Process is extremely complex, needs repeatedly to switch between more special instruments, take time and effort, and from collecting, sample is past to testing result is taken
Toward the time of the upper a couple of days such as needs.And entirely instrument is very huge, heavy, needs to be carried out by professional under private environment
Operation is safeguarded also not convenient.
Therefore, how a kind of full-automatic integrated specimen preparation system is provided, its acceptable biological sample input is made, and
Amplified production is directly exported, operating process does not depend on other laboratory equipments, and operating process is simple, and operating personnel are wanted in reduction
It asks, becomes those skilled in the art's important technological problems urgently to be resolved hurrily.
Invention content
In view of this, first of the present invention is designed to provide a kind of full-automatic sample preparation microfluidic system, so that
Its acceptable biological sample input, and amplified production is directly exported, operating process does not depend on other laboratory equipments, operating process
Simply, it reduces and operating personnel is required；Second object of the present invention is to provide a kind of above-mentioned full-automatic sample preparation miniflow
The preparation method and application method of control system.
To achieve the above object, the present invention provides the following technical solutions：
A kind of full-automatic sample preparation microfluidic system, including：
Chip body, be disposed with thereon by the concatenated entrance of Micro-flow pipe, the first chip valve, amplification chamber,
Second chip valve, product delivery outlet and waste liquid delivery outlet；
Sample interface module is tightly connected with the chip body, and the sample interface module includes from top to bottom successively
The lysate storage pool and amplifing reagent storage pool of setting, between the lysate storage pool and the amplifing reagent storage pool
It is sealed off by the first sealed compartment, the lysate storage pool is tried in the first sealed compartment breakage with the amplification
Agent storage pool is connected to, and lower end and the entrance of the amplifing reagent storage pool are sealed off by the second sealed compartment, described
The lower end of amplifing reagent storage pool is connected in the second sealed compartment breakage with the entrance.
Preferably, the sample interface module includes upper layer module and lower module, and the lysate storage pool opens up
In the upper layer module, the amplifing reagent storage pool is opened in the lower module, the upper layer module and lower layer's mould
Block is tightly connected.
Preferably, the amplifing reagent storage pool is connected to by gas vent with outside.
Preferably, the gas vent is opened in the upper layer module.
Preferably, be provided on the side wall of the amplifing reagent storage pool connect with the lysate storage pool for breaking
The flow-guiding structure of bad surface tension of liquid.
Preferably, the flow-guiding structure is the water conservancy diversion being opened in up and down on the side wall of the amplifing reagent storage pool
Slot.
Preferably, first sealed compartment and second sealed compartment are made of fragile material.
Preferably, the product delivery outlet is identical as the amplification chamber volume.
A kind of preparation method of full-automatic sample preparation microfluidic system, including step：
A1 sample interface module) is sealedly attached to chip body；
A2 amplifing reagent system solution) is added to the amplifing reagent storage pool of sample interface module, then to amplifing reagent
System solution is dried to form amplification system powder；
A3) lysate is added in the lysate storage pool of sample interface module；
A4) each interface on sample interface module and chip body is closed.
Preferably, the sample interface module includes upper layer module and lower module, the step a1) it is specially by sample
The lower module of product interface module is sealedly attached to chip body, subsequently into the step a2).
Preferably, the step a3) be specially：
A3.1 upper layer module) is sealedly attached to lower module, makes the first sealed compartment by lysate storage pool and amplification
Reagent storage pool is isolated；
A3.2) lysate is added in the lysate storage pool of sample interface module.
Preferably, into the step a3) before, break between amplifing reagent storage pool and the entrance of chip body
Two sealed compartments make the two be connected to.
A kind of application method of full-automatic sample preparation microfluidic system, including step：
B1 sample) is extracted, and is rinsed in the lysate of lysate storage pool, sample DNA molecule is discharged into lysate
In；
B2 it) if the second sealed compartment is broken in the preparation process of full-automatic sample preparation microfluidic system, closes
After first chip valve, then the first sealed compartment is broken, the lysate containing sample DNA molecule is made to enter in amplifing reagent storage pool
Dissolving amplification system powder forms complete PCR amplification system；If the second sealed compartment is in the micro-fluidic system of full-automatic sample preparation
It is not broken in the preparation process of system, then breaks the first sealed compartment, the lysate containing sample DNA molecule is made to enter amplification examination
Dissolving amplification system powder is formed in agent storage pool breaks the second sealed compartment again after complete PCR amplification system；
B3) negative pressure source is connected in waste liquid equipped at outlet port and open the first chip valve and the second chip valve, by PCR amplification body
System is drained to amplification chamber；
B4 the first chip valve and the second chip valve) are closed, PCR amplification system is made to carry out temperature cycles expansion in amplification chamber
Increase；
B5 after the completion of) expanding, the first chip valve and the second chip valve is opened, amplified production is drained to product delivery outlet
To extract；
B6 the waste liquid after amplified production) is extracted to export through waste liquid delivery outlet.
To achieve the above object, full-automatic sample preparation microfluidic system provided by the invention, including chip body and
Sample interface module, wherein be disposed in chip body through the concatenated entrance of Micro-flow pipe, the first chip valve, expanded
Increase chamber, the second chip valve, product delivery outlet and waste liquid delivery outlet；Sample interface module is tightly connected with chip body, sample
This interface module includes the lysate storage pool set gradually from top to bottom and amplifing reagent storage pool, lysate storage pool with
Between amplifing reagent storage pool by the first sealed compartment seal off, lysate storage pool in the first sealed compartment breakage with
Amplifing reagent storage pool be connected to so that lysate can enter amplifing reagent storage pool in, the lower end of amplifing reagent storage pool with
Entrance is sealed off by the second sealed compartment, and the lower end of amplifing reagent storage pool connects in the second sealed compartment breakage with entrance
It is logical；
When preparing above-mentioned full-automatic sample preparation microfluidic system, sample interface module is sealedly attached to chip first
Main body；Then amplifing reagent system solution is added to the amplifing reagent storage pool of sample interface module, and to amplifing reagent system
Solution is dried to form amplification system powder, and before amplifing reagent system solution is dried and to form amplification system powder,
Ensure to be seal isolation state between amplifing reagent storage pool and the entrance of chip sample；Thereafter make lysate storage pool and amplification
Reagent storage pool is in seal isolation state, and lysate is added in the lysate storage pool of sample interface module；Finally will
Each interface on sample interface module and chip body is closed to store and to transport；
In use, first opening the sealing of lysate storage pool, sample is extracted by swab stick or other sampling equipments, and
It is rinsed in the lysate of lysate storage pool, sample DNA molecule is discharged into lysate；Thereafter, if the second sealed compartment
It is broken in the preparation process of full-automatic sample preparation microfluidic system, then to avoid lysate that from cannot fully dissolving amplification body
The case where being powder, after should then closing the first chip valve, then breaks the first sealed compartment, makes splitting containing sample DNA molecule
Solution liquid enters dissolving amplification system powder in amplifing reagent storage pool and forms complete PCR amplification system, utilizes the surface of liquid
Air pressure in tension and Micro-flow pipe avoids lysate from being directly entered Micro-flow pipe；If the second sealed compartment is full-automatic
It is not broken in the preparation process of sample preparation microfluidic system, then breaks the first sealed compartment, made containing sample DNA molecule
Lysate, which enters in amplifing reagent storage pool, to be dissolved amplification system powder and forms after complete PCR amplification system that break second again close
Sealing layer；After lysate and amplification system powder are mutually dissolved, the first chip valve and the second chip valve are opened, and in waste liquid
Equipped at outlet port connects negative pressure source, and PCR amplification system is drained to amplification chamber；After PCR amplification system reaches amplification chamber, close
The first chip valve and the second chip valve are closed, PCR amplification system is made to carry out temperature cycles amplification in amplification chamber；Amplification is completed
Afterwards, the first chip valve and the second chip valve are opened, amplified production is drained to product delivery outlet to extract；
It can be seen that above-mentioned full-automatic sample preparation microfluidic system, simple in structure, convenient for preparing, and have unique
It is loaded module and liquid storage pattern, can the biological samples such as buccal swab, saliva, cast-off cells be carried out with the automation of integrated form
Processing, directly gives amplified production, and whole process is completely without with the additional laboratory such as pipettor, shaker, centrifuge
Special equipment or instrument so that the preparation flow can be detached from laboratory environment progress, easy to operate, can be by slightly training
Layman completes, and makes it possible the Site Detection of DNA.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Obtain other attached drawings according to these attached drawings.
Fig. 1 is the axonometric drawing of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention；
Fig. 2 is the vertical view of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention；
Fig. 3 is the axonometric drawing of the upper layer module of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention；
Fig. 4 is the axonometric drawing of the lower module of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention；
Fig. 5 is the preparation process chips semi-finished product of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention
Sectional view；
Fig. 6 is the sectional view that the preparation of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention is completed；
Fig. 7 is full-automatic sample preparation microfluidic system provided in an embodiment of the present invention in the sectional view being loaded when operating；
Fig. 8 is cuing open when full-automatic sample preparation microfluidic system provided in an embodiment of the present invention forms PCR amplification system
View；
Fig. 9 is section view of the full-automatic sample preparation microfluidic system provided in an embodiment of the present invention when carrying out PCR amplification
Figure.
In figure：
1 is upper layer module；2 be lower module；3 be lysate storage pool；4 be gas vent；5 be amplifing reagent storage pool；6
For flow-guiding structure；7 be chip body；8 be amplification chamber；9 be the first chip valve；10 be product delivery outlet；11 export for waste liquid
Mouthful；12 be entrance；13 be Micro-flow pipe；14 be the second chip valve；15 be the second sealed compartment；16 be amplification system powder；
17 be lysate；18 be the first sealed compartment；19 be swab stick.
Specific implementation mode
First of the present invention is designed to provide a kind of full-automatic sample preparation microfluidic system, this is used for full-automatic sample
The structure design of this preparation microfluidic system makes its acceptable biological sample input, and directly exports amplified production, operating process
Other laboratory equipments are not depended on, operating process is simple, reduces and requires operating personnel；Second object of the present invention is to carry
For a kind of preparation method and application method of above-mentioned full-automatic sample preparation microfluidic system.
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
It please refers to Fig.1 and Fig. 2, Fig. 1 surveys for the axis of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention
Figure, Fig. 2 are the vertical view of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention.
A kind of full-automatic sample preparation microfluidic system provided in an embodiment of the present invention, including chip body 7 and sample
Interface module.
Wherein, it is disposed with through 13 concatenated entrance 12 of Micro-flow pipe, the first chip valve 9, expands in chip body 7
Increasing chamber 8, the second chip valve 14, product delivery outlet 10 and waste liquid delivery outlet 11,11 rear end of waste liquid delivery outlet connects negative pressure source,
Such as precise injection pump or peristaltic pump be with the liquid position in accurate control Micro-flow pipe 13, above-mentioned product delivery outlet 10 with
And waste liquid delivery outlet 11 is during the entire process of chip operates, above opening sealed by adhesive tape bonding, when use again
It opens；Sample interface module and chip body 7 are tightly connected, the sealed connection can be fluid sealant bonding, ultrasonic welding or
It is the forms such as heat sealing, sample interface module includes that the lysate storage pool 3 set gradually from top to bottom and amplifing reagent are deposited
Reservoir 5 is sealed off between lysate storage pool 3 and amplifing reagent storage pool 5 by the first sealed compartment 18, lysate storage
Pond 3 is connected to when the first sealed compartment 18 is damaged with amplifing reagent storage pool 5, so that lysate 17 can be deposited into amplifing reagent
In reservoir 5, lower end and the entrance 12 of amplifing reagent storage pool 5 are sealed off by the second sealed compartment 15, amplifing reagent storage
The lower end in pond 5 is connected to when the second sealed compartment 15 is damaged with entrance 12；First sealed compartment 18 and the second sealed compartment 15
It is all made of the making of rapid wear material；First chip valve 9 and the second chip valve 14 can be existing arbitrary chip valve arrangements, such as pass through
Common PDMS valve bodies etc. in the pneumatic operated valve or micro-fluidic chip of allusion quotation.
A plurality of Micro-flow pipe 13 can be set in the chip body 7 of each full-automatic sample preparation microfluidic system, and
The upstream of every miniflow keyholed back plate is respectively provided with sample interface module, so that above-mentioned full-automatic sample preparation microfluidic system can be simultaneously
Multiple samples are detected, raising efficiency, in the embodiment that the present invention illustrates, full-automatic sample preparation microfluidic system includes
Two sample interface modules and two Micro-flow pipes 13 being attached thereto respectively.
Compared with prior art, the full-automatic sample preparation microfluidic system detected provided by the present invention for live DNA,
Amplification system powder 16 is stored when preparation in amplifing reagent storage pool 5, lysate 17 is stored in lysate storage pool 3, and expand
Increase and be isolated by delicate first sealed compartment 18 between reagent storage pool 5 and lysate storage pool 3, when in use, by sample
This mixes with lysate 17, so that sample DNA molecule is discharged into lysate 17, then breaks the first sealed compartment 18, make containing
The lysate 17 of sample DNA molecule enters dissolving amplification system powder 16 in amplifing reagent storage pool 5 and forms PCR amplification system,
And in the process, ensured at the PCR amplification system to be formed using the second sealed compartment 15 or surface tension of liquid and air pressure
In amplifing reagent storage pool 5, the entrance 12 without entering chip body；It is then turned on the first chip valve 9 and the second chip
Valve 14, and negative pressure source is connected at waste liquid delivery outlet 11, PCR amplification system is drained to amplification chamber 8；Work as PCR amplification system
After reaching amplification chamber 8, the first chip valve 9 and the second chip valve 14 are closed, PCR amplification system is made to be carried out in amplification chamber 8
Temperature cycles expand；After the completion of amplification, the first chip valve 9 and the second chip valve 14 are opened, amplified production is drained to product
Delivery outlet 10 is to extract；
It can be seen that above-mentioned full-automatic sample preparation microfluidic system, simple in structure, convenient for preparing, and have unique
It is loaded module and liquid storage pattern, can the biological samples such as buccal swab, saliva, cast-off cells be carried out with the automation of integrated form
Processing, directly gives amplified production, and whole process is completely without with the additional laboratory such as pipettor, shaker, centrifuge
Special equipment or instrument so that the preparation flow can be detached from laboratory environment progress, easy to operate, can be by slightly training
Layman completes, and makes it possible the Site Detection of DNA.
Amplification system powder 16 can directly make an addition in amplifing reagent storage pool 5, and it is molten also amplifing reagent system can be first added
Then liquid is dried it to form amplification system powder 16, the drying of amplifing reagent system solution can be in vacuum drying chamber
Or carried out in freeze dryer, in embodiments of the present invention, expanded by the way of amplifing reagent system solution and then drying is added
Increasing system powder 16, for ease of operation, sample interface module uses the structure of levels, including upper layer module 1 and lower layer's mould
Block 2, lysate storage pool 3 are opened in upper layer module 1, and amplifing reagent storage pool 5 is opened in lower module 2, and upper layer module 1 is under
Layer module 2 is tightly connected, can be used between upper layer module 1 and lower module 2 the good double faced adhesive tape of bio-compatibility be adhesively fixed or
Person is sealedly connected and fixed using other modes.
In this way, in the preparation, can lower module 2 be first fixed on chip body, amplifing reagent system solution is added, it is dry
Amplification system powder 16 is formed in amplifing reagent storage pool 5 afterwards, then upper layer module 1 is connected on lower module 2 again, into
The operation of row next step.
In above-mentioned drying process, due to the effect of surface tension, amplification system powder 16 can be evenly distributed in amplification
5 bottom margin area of reagent storage pool, as shown in figure 5, therefore amplification system powder 16 can obtained by the second sealed compartment 15
It breaks to facilitate the operation during genetic test, it is of course also possible to retain the second sealed compartment 15, is grasped in genetic test below
Row destroys again when making.In embodiments of the present invention, it is the operating process for simplifying during genetic test, is obtained in preparation process
The second sealed compartment 15 is destroyed after amplification system powder 16.
It is destroyed in preparation process since the second sealed compartment 15 is destroyed, in application process, lysate
During 17 are mixed to form PCR amplification system solution into amplifing reagent storage pool 5 with amplification system powder 16, it can only rely on
Micro-flow pipe 13 and the surface tension of liquid ensure that PCR amplification system solution is in amplifing reagent storage pool 5 without in advance
Into in Micro-flow pipe 13, to ensure abundant dissolving of the lysate 17 to amplification system powder 16, but due to amplifing reagent
Storage pool 5 arranges that lysate 17 is flowed into from top can lead to 5 air pressure liter of amplifing reagent storage pool with about 3 lysate storage pool
Height, the surface tension for being easy to cause liquid and support of the air pressure to PCR amplification system solution in Micro-flow pipe 13, to make to split
It solves liquid 17 and enters Micro-flow pipe 13 too early, to solve the above problems, in embodiments of the present invention, amplifing reagent storage pool 5 is logical
It crosses gas vent 4 to be connected to outside, as shown in figures 1 and 3, further, gas vent 4 is opened in upper layer module 1, and along thickness
Upper layer module 1 is run through in direction.Certainly, gas vent 4 open up position and shape is not unique, for example, it can be opened in upper layer
In at least one of module 1 and the mating surface of lower module 2, it can also be opened in upper layer module 1 but be L-shaped etc., only
Want can to realize the exhaust of amplifing reagent storage pool 5.
When assembling upper layer module 1 and lower module 2, it must ensure that lysate storage pool 3 and gas vent 4 can be with
Amplifing reagent storage pool 5 communicates.
Above-mentioned technical proposal is advanced optimized, as shown in figures 1 to 6, sectional dimension is gradually from top to bottom for lysate storage pool 3
Contracting, big opening end are opened in the top of upper layer module 1, and osculum end is opened in the bottom of upper layer module 1.Further, such as Fig. 6
Shown, the longitudinal section of lysate storage pool 3 is right-angled trapezium.
First sealed compartment 18 is arranged in the bottom of upper layer module 1 to seal the osculum end of lysate storage pool 3.
Since the lower ending opening of lysate storage pool 3 is smaller, in application process, when the first sealed compartment 18 is destroyed
When, it is easy the effect due to surface tension of liquid, causes lysate 17 that can not enter amplifing reagent under the effect of gravity
Storage pool 5, for this purpose, in embodiments of the present invention, being provided on the side wall of amplifing reagent storage pool 5 and 3 phase of lysate storage pool
The flow-guiding structure 6 for destroying surface tension of liquid connect is being split in use, destroying lysate 17 by the flow-guiding structure 6
The liquid level tension for solving 3 osculum end of liquid storage pool, can smoothly flow into amplifing reagent storage pool 5, flow-guiding structure 6 can be used
Various ways realize, for example, can make the partial sidewall that amplifing reagent storage pool 5 connects with lysate storage pool 3 roughness and its
His part is different, alternatively, as shown in figure 4, in a kind of specific embodiment of the present invention, flow-guiding structure 6 is to open up up and down
Diversion trench on the side wall of amplifing reagent storage pool 5, the diversion trench be provided with it is multiple, in the side wall of amplifing reagent storage pool 5
Upper parallel arrangement.
In embodiments of the present invention, the first sealed compartment 18 and the second sealed compartment 15 are made of fragile material, crisp
Property material include but not limited to aluminium, copper, plastics etc., i.e. the first sealed compartment 18 and the second sealed compartment 15 can be aluminium foil,
Any one in copper foil and plastic film, and fragile material used in the first sealed compartment 18 and the second sealed compartment 15
Can be consistent, or can respectively use different materials according to use demand.
Above-mentioned technical proposal is advanced optimized, in embodiments of the present invention, for ease of the amplification production formed in amplification chamber 8
Object all exports, and product delivery outlet 10 is identical as amplification 8 volume of chamber.
The embodiment of the present invention additionally provides a kind of method preparing above-mentioned full-automatic sample preparation microfluidic system, this method
Including step：
a1：Sample interface module is sealedly attached to chip body 7；,
It must be tightly connected between sample interface module and chip body 7 to avoid leakage, specifically, sealed connection can adopt
With forms such as sealing glue sticking, ultrasonic welding or heat sealings, step a2 and step a3 below can be in sample interface moulds
Block is completed before being sealedly attached in chip body 7, can also be complete after sample interface module is connected in chip body 7
At in the embodiment of the present invention, step a2 and step a3 are carried out after sample interface module is sealedly attached to chip body 7.
a2：Amplifing reagent system solution is added to the amplifing reagent storage pool 5 of sample interface module, then to amplifing reagent
System solution is dried to form amplification system powder 16；
During step a2 is carried out, ensure that amplifing reagent system solution exists only in amplifing reagent storage pool 5, therefore answer
It is sealed when to 5 lower end of amplifing reagent storage pool, the entrance 12 of itself and chip body 7 is sealed off.
a3：Lysate 17 is added in the lysate storage pool 3 of sample interface module；
After the above step is finished, cracking is added into the lysate storage pool 3 sealed off with amplifing reagent storage pool 5
Liquid 17, since the full-automatic sample preparation microfluidic system in this case is mainly for the cracking of human body cell, for human body cell,
Alkaline condition can preferably make membranolysis released dna molecule, and therefore, lysate 17 herein is alkaline bleach liquor cleavage liquid 17,
Make cell cracking using pH conditions；Certainly, above-mentioned alkaline bleach liquor cleavage liquid 17 is only the present invention according to full-automatic sample preparation miniflow
The preferred embodiment that the main application of control system is made, is actually not limited thereto, those skilled in the art according to it is complete from
The difference of dynamic sample preparation microfluidic system purposes, can also be added other types of lysate 17, not limit herein.
a4：By each interface closing on sample interface module and chip body 7.
Wait for that step a3 is completed, it, can be directly by full-automatic sample preparation microfluidic system after the storage in place of lysate 17
It is stored and is transported after total interface sealing.Sealing outer packing is removed again using preceding.
Above-mentioned technical proposal is advanced optimized, for ease of the preparation of full-automatic sample preparation microfluidic system, in the present invention
In embodiment, as shown in Figure 1, sample interface module uses double-layer structure, including upper layer module 1 and lower module 2, because
This, the addition of amplifing reagent system solution can carry out in two modules respectively with dry and lysate 17 sectional drawing, mutually not
It influences, on this basis, step a1 is specially that the lower module 2 of sample interface module is sealedly attached to chip body 7, then
Enter step a2, the sectional view of chip semi-finished product is formed by after the completion of step a2 as shown in figure 5, it can be seen from the figure that by
The influence of surface tension of liquid, amplification system powder 16 are evenly distributed on 5 bottom margin area of amplifing reagent storage pool, and will be by
Second sealed compartment, 15 closed 5 bottom centre of amplifing reagent storage pool vacates, at this point, for ease of later application, simplifies behaviour
Make flow, before entering step a3, breaks the second sealing between amplifing reagent storage pool 5 and the entrance 12 of chip body 7
Interlayer 15 makes the two be connected to.
In embodiment as shown in FIG. 6, step a3 is specially：
a3.1：Upper layer module 1 is sealedly attached to lower module 2, make the first sealed compartment 18 by lysate storage pool 3 and
Amplifing reagent storage pool 5 is isolated；
Upper layer module 1 can be adhesively fixed with lower module 2 by the good double faced adhesive tape of bio-compatibility, naturally it is also possible to be passed through
Other modes are fixed, and the bottom of upper layer module 1 is provided with the first sealed compartment 18 with by lysate storage pool 3 and amplifing reagent
Storage pool 5 is isolated.
a3.2：Lysate 17 is added in the lysate storage pool 3 of sample interface module.
Fig. 7-Fig. 9 is please referred to, Fig. 7 is that full-automatic sample preparation microfluidic system provided in an embodiment of the present invention is grasped in sample-adding
Sectional view when making, when Fig. 8 is that full-automatic sample preparation microfluidic system provided in an embodiment of the present invention forms PCR amplification system
Sectional view, Fig. 9 be section view of the full-automatic sample preparation microfluidic system provided in an embodiment of the present invention when carrying out PCR amplification
Figure, the embodiment of the present invention additionally provide a kind of application method of full-automatic sample preparation microfluidic system, including step：
b1：Sample is extracted, and is rinsed in the lysate of lysate storage pool 3 17, sample DNA molecule is discharged into cracking
In liquid 17；
In the illustrated embodiment, sample is extracted using swab stick 19, as shown in fig. 7, swab stick 19 is stretched into after dipping sample
It is rinsed in lysate storage pool 3, the sample cell being attached in swab stick 19 during rinsing will fall off or rupture
To which sample DNA molecule is discharged into lysate 17.
b2：If the second sealed compartment 15 is broken in the preparation process of full-automatic sample preparation microfluidic system, close
After closing the first chip valve 9, then the first sealed compartment 18 is broken, the lysate 17 containing sample DNA molecule is made to enter amplifing reagent
Amplification system powder 16 is dissolved in storage pool 5 forms complete PCR amplification system；If the second sealed compartment 15 is in full-automatic sample
It prepares and is not broken in the preparation process of microfluidic system, then break the first sealed compartment 18, make splitting containing sample DNA molecule
Solution liquid 17 enters dissolving amplification system powder 16 in amplifing reagent storage pool 5 and formed after complete PCR amplification system breaks the again
Two sealed compartments 15；
After rinsing, swab stick 19 is pressed into 3 bottom of lysate storage pool down, the first sealed compartment 18 is punctured, later
Swab stick 19 as consumptive material can abandon.After first sealed compartment 18 is punctured, lysate 17 can be transported downwards due to gravity
It moves into amplifing reagent storage pool 5, in the process, as shown in fig. 7, the second sealed compartment 15 is broken in preparation process
It is bad, therefore to make lysate 17 flow downward process control, must meet jointly there are three condition：First, the first chip valve 9 closes
It closes, blocks Micro-flow pipe 13 to prevent liquid from entering 13 entrance 12 of Micro-flow pipe；Secondly, exhaust outlet is stored with amplifing reagent
Pond 5 is interconnected to ensure that amplifing reagent storage pool 5 is balanced with external pressure；Finally, the lower end interface of lysate 17 and water conservancy diversion
Structure 6 is contacted to overcome the surface tension of its lower surface；On the basis of three conditions meet herein, lysate 17 can be along water conservancy diversion knot
Structure 6 flows downward into amplifing reagent storage pool 5, since rear end the first chip valve 9 is closed, the sky in amplifing reagent storage pool 5
Gas is discharged by exhaust outlet, and the surface tension of 17 bottom of lysate and the air pressure inside Micro-flow pipe 13 can be maintained lysate
17 bottom surface makes it not enter entrance 12.
Certainly, the above only preferred embodiment provided by the invention, in fact, there is also the second sealed compartments 15
The case where not being destroyed in preparation process, in the case, since the second sealed compartment 15 can still play seal isolation
Effect, therefore the first chip valve 9 close whether it is not important, after the completion of can waiting for that lysate 17 is mixed with amplification system powder 16
Directly destroy the second sealed compartment 15.
b3：Negative pressure source is connected at waste liquid delivery outlet 11 and opens the first chip valve 9 and the second chip valve 14, by PCR
Amplification system is drained to amplification chamber 8；
After the lysate 17 in step b2 is by amplification system powder 16 fully dissolving, using negative at waste liquid delivery outlet 11
PCR amplification system solution is drained to amplification chamber 8 to carry out amplification operation by potential source vacuum suction.
b4：The first chip valve 9 and the second chip valve 14 are closed, PCR amplification system is made to be followed into trip temperature in amplification chamber 8
Circle amplification；
First chip valve 9 and the second chip valve 14 are closed to ensure to expand the leakproofness of chamber 8, when to avoid high temperature
The bubble expansion generated in amplification chamber 8, then installation heat source and low-temperature receiver are molten to PCR amplification system outside amplification chamber 8
Liquid carries out temperature cycles.
b5：After the completion of amplification, the first chip valve 9 and the second chip valve 14 are opened, it is defeated that amplified production is drained to product
Outlet 10 is to extract；
b6：The waste liquid after amplified production is extracted to export through waste liquid delivery outlet.
Full-automatic sample preparation microfluidic system provided in an embodiment of the present invention and preparation method thereof and application method, have
Following advantages：1, easy to operate, do not need the laboratories special equipment such as pipettor, centrifuge and instrument can be realized it is fully integrated from
It is prepared by dynamicization DNA sample；2, efficient, cell cracking, DNA extractions are carried out on chip automatically with expanding, and whole flow process can be with
It is completed within two hours；3, popular, it is easy to operate so that also being capable of operation instrument without the general public of professional training
Device completes sample preparation procedures, and facility is provided for the popularization and promotion of gene technology.
Each embodiment is described by the way of progressive in this specification, the highlights of each of the examples are with other
The difference of embodiment, just to refer each other for identical similar portion between each embodiment.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest range caused.
Claims (14)
1. a kind of full-automatic sample preparation microfluidic system, which is characterized in that including：
Chip body is disposed with thereon through the concatenated entrance of Micro-flow pipe, the first chip valve, amplification chamber, second
Chip valve, product delivery outlet and waste liquid delivery outlet；
Sample interface module is tightly connected with the chip body, and the sample interface module includes setting gradually from top to bottom
Lysate storage pool and amplifing reagent storage pool, pass through between the lysate storage pool and the amplifing reagent storage pool
First sealed compartment seals off, and the lysate storage pool is deposited in the first sealed compartment breakage with the amplifing reagent
Reservoir is connected to, and lower end and the entrance of the amplifing reagent storage pool are sealed off by the second sealed compartment, the amplification
The lower end of reagent storage pool is connected in the second sealed compartment breakage with the entrance.
2. full-automatic sample preparation microfluidic system according to claim 1, which is characterized in that the sample interface module
Including upper layer module and lower module, the lysate storage pool is opened in the upper layer module, the amplifing reagent storage
Pond is opened in the lower module, and the upper layer module is tightly connected with the lower module.
3. full-automatic sample preparation microfluidic system according to claim 2, which is characterized in that the amplifing reagent storage
Pond is connected to by gas vent with outside.
4. full-automatic sample preparation microfluidic system according to claim 3, which is characterized in that the gas vent is opened in
The upper layer module.
5. full-automatic sample preparation microfluidic system according to any one of claims 1-4, which is characterized in that described to split
Solving liquid storage pool, sectional dimension is tapered from top to bottom.
6. full-automatic sample preparation microfluidic system according to any one of claims 1-4, which is characterized in that the expansion
Increase the water conservancy diversion for destroying surface tension of liquid for being provided on the side wall of reagent storage pool and connecting with the lysate storage pool
Structure.
7. full-automatic sample preparation microfluidic system according to claim 6, which is characterized in that the flow-guiding structure is upper
The diversion trench being opened in lower perforation on the side wall of the amplifing reagent storage pool.
8. full-automatic sample preparation microfluidic system according to claim 1, which is characterized in that first sealed compartment
And second sealed compartment is made of fragile material.
9. full-automatic sample preparation microfluidic system according to claim 1, which is characterized in that the product delivery outlet with
The amplification chamber volume is identical.
10. a kind of preparation method of full-automatic sample preparation microfluidic system, which is characterized in that including step：
A1 sample interface module) is sealedly attached to chip body；
A2 amplifing reagent system solution) is added to the amplifing reagent storage pool of sample interface module, then to amplifing reagent system
Solution is dried to form amplification system powder, and before amplifing reagent system solution is dried and to form amplification system powder,
Ensure to be seal isolation state between amplifing reagent storage pool and the entrance of chip sample；
A3 so that lysate storage pool is in seal isolation state with amplifing reagent storage pool, lysate is added to sample interface
In the lysate storage pool of module；
A4) each interface on sample interface module and chip body is closed.
11. the preparation method of full-automatic sample preparation microfluidic system according to claim 10, which is characterized in that described
Sample interface module includes upper layer module and lower module, the step a1) it is specially by lower layer's mould of sample interface module
Block is sealedly attached to chip body, subsequently into the step a2).
12. the preparation method of full-automatic sample preparation microfluidic system according to claim 11, which is characterized in that described
Step a3) be specially：
A3.1 upper layer module) is sealedly attached to lower module, makes the first sealed compartment by lysate storage pool and amplifing reagent
Storage pool is isolated；
A3.2) lysate is added in the lysate storage pool of sample interface module.
13. the preparation method of the full-automatic sample preparation microfluidic system according to claim 10-12 any one, special
Sign is, into the step a3) before, break between amplifing reagent storage pool and the entrance of chip body second sealing every
Layer makes the two be connected to.
14. a kind of application method of full-automatic sample preparation microfluidic system, which is characterized in that including step：
B1 sample) is extracted, and is rinsed in the lysate of lysate storage pool, sample DNA molecule is discharged into lysate；
B2) if the second sealed compartment is broken in the preparation process of full-automatic sample preparation microfluidic system, first is closed
After chip valve, then the first sealed compartment is broken, so that the lysate containing sample DNA molecule is entered in amplifing reagent storage pool and dissolve
Amplification system powder forms complete PCR amplification system；If the second sealed compartment is in full-automatic sample preparation microfluidic system
It is not broken in preparation process, then breaks the first sealed compartment, so that the lysate containing sample DNA molecule is entered amplifing reagent and deposit
Dissolving amplification system powder is formed in reservoir breaks the second sealed compartment again after complete PCR amplification system；
B3) negative pressure source being connected in waste liquid equipped at outlet port and opening the first chip valve and the second chip valve, PCR amplification system is drawn
It flow to amplification chamber；
B4 the first chip valve and the second chip valve) are closed, PCR amplification system is made to carry out temperature cycles amplification in amplification chamber；
B5 after the completion of) expanding, the first chip valve and the second chip valve are opened, by amplified production be drained to product delivery outlet with into
Row extraction；
B6 the waste liquid after amplified production) is extracted to export through waste liquid delivery outlet.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112322453A (en) * | 2020-12-03 | 2021-02-05 | 中国计量科学研究院 | Micro-fluidic chip for nucleic acid extraction, amplification and detection |
| CN112538425A (en) * | 2020-10-23 | 2021-03-23 | 北京理工大学 | On-chip nucleic acid amplification detection system and method based on micro-fluidic chip |
| WO2021237397A1 (en) * | 2020-05-25 | 2021-12-02 | 杭州梓晶生物有限公司 | Fluid control device and fluid control method |
| CN114405566A (en) * | 2022-02-08 | 2022-04-29 | 江苏液滴逻辑生物技术有限公司 | Freeze-drying ball pre-embedded structure, digital micro-fluidic chip and pre-embedded liquid injection method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110124049A1 (en) * | 2007-08-07 | 2011-05-26 | Mo-Huang Li | Integrated microfluidic device for gene synthesis |
| US20140194313A1 (en) * | 2011-06-06 | 2014-07-10 | Cornell University | Microfluidic device for extracting, isolating, and analyzing dna from cells |
| CN104946510A (en) * | 2014-03-31 | 2015-09-30 | 博奥生物集团有限公司 | Nucleic acid amplification/microarray detection integrated microfluidic device |
| CN105277725A (en) * | 2014-07-01 | 2016-01-27 | 清华大学 | Integrated micro-fluidic system for nucleic acid analysis and detection |
| CN206375900U (en) * | 2016-12-14 | 2017-08-04 | 深圳市埃克特生物科技有限公司 | A kind of miniflow bag for nucleic acid integration detection |
| CN107893020A (en) * | 2017-11-27 | 2018-04-10 | 深圳华炎微测医疗科技有限公司 | Molecule diagnoses micro-fluidic chip and molecule diagnosis micro-fluidic chip system and their application |
-
2018
- 2018-05-23 CN CN201810502150.0A patent/CN108728327B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110124049A1 (en) * | 2007-08-07 | 2011-05-26 | Mo-Huang Li | Integrated microfluidic device for gene synthesis |
| US20140194313A1 (en) * | 2011-06-06 | 2014-07-10 | Cornell University | Microfluidic device for extracting, isolating, and analyzing dna from cells |
| CN104946510A (en) * | 2014-03-31 | 2015-09-30 | 博奥生物集团有限公司 | Nucleic acid amplification/microarray detection integrated microfluidic device |
| CN105277725A (en) * | 2014-07-01 | 2016-01-27 | 清华大学 | Integrated micro-fluidic system for nucleic acid analysis and detection |
| CN206375900U (en) * | 2016-12-14 | 2017-08-04 | 深圳市埃克特生物科技有限公司 | A kind of miniflow bag for nucleic acid integration detection |
| CN107893020A (en) * | 2017-11-27 | 2018-04-10 | 深圳华炎微测医疗科技有限公司 | Molecule diagnoses micro-fluidic chip and molecule diagnosis micro-fluidic chip system and their application |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021237397A1 (en) * | 2020-05-25 | 2021-12-02 | 杭州梓晶生物有限公司 | Fluid control device and fluid control method |
| CN112538425A (en) * | 2020-10-23 | 2021-03-23 | 北京理工大学 | On-chip nucleic acid amplification detection system and method based on micro-fluidic chip |
| CN112322453A (en) * | 2020-12-03 | 2021-02-05 | 中国计量科学研究院 | Micro-fluidic chip for nucleic acid extraction, amplification and detection |
| CN112322453B (en) * | 2020-12-03 | 2023-04-11 | 中国计量科学研究院 | Micro-fluidic chip for nucleic acid extraction, amplification and detection |
| CN114405566A (en) * | 2022-02-08 | 2022-04-29 | 江苏液滴逻辑生物技术有限公司 | Freeze-drying ball pre-embedded structure, digital micro-fluidic chip and pre-embedded liquid injection method |
| CN114405566B (en) * | 2022-02-08 | 2022-12-02 | 江苏液滴逻辑生物技术有限公司 | Freeze-drying ball pre-embedded structure, digital micro-fluidic chip and pre-embedded liquid injection method |
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