CN109746062A - Microlayer model generating means - Google Patents
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- CN109746062A CN109746062A CN201711074985.2A CN201711074985A CN109746062A CN 109746062 A CN109746062 A CN 109746062A CN 201711074985 A CN201711074985 A CN 201711074985A CN 109746062 A CN109746062 A CN 109746062A
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Abstract
The present invention provides a kind of microlayer model generating means, and the microlayer model generating means include the first component and second component, and the first component is fixedly connected with second component；The first component is that microlayer model generates chip, the generation for microlayer model；It is microlayer model sample pipetting volume and generation microlayer model collection device, the collection of the microlayer model of the sample-adding and generation of the oily phase sample and aqueous sample for the first component with the second component.Microlayer model generating means of the invention can quick, reliable, parallel generation is uniform micron dimension " Water-In-Oil "/" oil-in-water " microlayer model, material and batch machining are low in cost；Sample injection, drop collection process are convenient, and whole process is not likely to produce cross contamination.
Description
Technical field
The present invention relates to microlayer model digital pcr technical fields, and in particular to a kind of microlayer model generating means.
Background technique
Microlayer model digital pcr technology (droplet digital PCR, ddPCR) is a kind of nucleic acid based on single-molecule PCR
Absolute quantification analysis technology.Just to become industry next for highly sensitive, high accuracy advantage with it for microlayer model digital pcr technology
Revolutionary technology.In recent years, with micro-nano manufacturing technology and micro-fluidic technologies (micro-nanofabrication and
Microfluidics development), microlayer model digital pcr technology encounter the best opportunity of break-through skill bottleneck.The technology by
Micro-fluidic chip, generating diameter is a few micrometers of drops for arriving hundreds of microns；Microlayer model wraps up unimolecule or unicellular, reaches reaction
It is totally-enclosed with detecting, it is fully integrated.Microlayer model digital pcr System Working Principle is: generating instrument by special microlayer model first will
Sample to be tested is assigned in " Water-In-Oil " microlayer model of a large amount of nanoliter levels (diameter is a few micrometers to hundreds of microns), the number of microlayer model
Amount is in million ranks.It is mutually isolated by oil reservoir between microlayer model since microlayer model quantity is enough, therefore each microlayer model is suitable
The DNA unimolecule of sample to be tested is contained only in one " microreactor ", microlayer model；Then, distinguish for these microlayer models
Pcr amplification reaction is carried out, and the fluorescence signal of drop is detected one by one by microlayer model analyzer, there is the micro- of fluorescence signal
Dripping interpretation is 1, and the droplet interpretation of fluorescence signal is not 0.Finally, according to Poisson distribution principle and the number of positive droplet with than
The target dna molecule number of example you can get it sample to be tested, realizes the absolute quantitation to sample of nucleic acid.
The committed step of microlayer model digital pcr technology is micron dimension " Water-In-Oil " quick, reliable, that parallel generation is uniform
Microlayer model.A core technology for generating microlayer model is the microlayer model generating means of design and processing based on microflow control technique.It is micro-
Drop formation device is widely used in clinical detection and needs to have following principle: (1) quickly, reliable, parallel generation is uniform
Micron dimension " Water-In-Oil "/" oil-in-water " microlayer model, the material and processing cost of the micro-fluid chip of (2) based on microflow control technique
Low, (3) operation is convenient, and (4) drop formation does not generate cross contamination during collecting.
Currently, the micro-fluidic chip based on dimethyl silicone polymer (PDMS) has been widely used for generating microlayer model.Firstly,
Researcher has the PDMS microlayer model chip of micron dimension using soft light carving technology (manual operation) processing.When PDMS microlayer model
After chip is successfully prepared, outlet being generated in its sample inlet, microlayer model and is punched using mechanical processing technique, assembly sample feeding pipe goes out
Sample pipe." oily phase " sample, " water phase " sample are drawn into syringe by manual mode.It then, will " oil by external injection pump
Phase " sample, " water phase " sample are by sample feeding pipe injection PDMS microlayer model chip.Finally, the microlayer model generated passes through sample outlet pipe
It is collected into routine experiment consumptive material, such as EP pipe.Although the research and development of PDMS microlayer model chip material are at low cost, laboratory processes work
Skill is simple, but its existing deficiency includes:
(1) PDMS is thermoelastic polymer material, such material is not suitable for technical grade injection molding, packaging technology.Add by hand
The PDMS microlayer model chip reliability of work is poor.
(2) PDMS microlayer model chip batch processing cost is high.
(3) injection of PDMS microlayer model chip sample, drop are collected as the cumbersome manual operation process of process, are unsuitable for clinic
Examine application.
(4) cross contamination is easy to produce during drop formation, collection.
For the deficiency of PDMS microlayer model chip, industry is for disadvantage mentioned above expansion research and development.The BioRad company in the U.S.
The microlayer model generating means based on polymer material have been developed with RainDance Technologies company.Biorad company
Microlayer model generating means, generation microlayer model needs be transferred in EP pipe from chip manually.The microlayer model of RainDance company
Generating means, oily phase sample need external instrument sample-adding, and with high costs.Existing for these microlayer model generating means itself not
Foot, limits it in the extensive use in clinical examination field.
Summary of the invention
For the deficiency of existing microlayer model generating means, the present invention provides a kind of microlayer model generation based on polymer material
Device.The characteristics of microlayer model generating means, is: (1) micron dimension " Water-In-Oil " quickly, reliable, parallel generation uniform or
" oil-in-water " microlayer model, (2) microlayer model chip is using thermoplastic material (such as makrolon material (PC), cyclic olefine copolymer
(COP) or polymethyl methacrylate material (PMMA), polypropylene (PP)), material and batch machining are low in cost, (3) by
In external pressure source, using the microlayer model generating means chip, sample injection, drop collection process become convenient, (4) integrated form
The design of microlayer model generating means, whole process are not likely to produce cross contamination.
In one embodiment, the present invention provides a kind of microlayer model generating means, and the microlayer model generating means include
The first component and second component, the first component are fixedly connected with second component；The first component is that microlayer model generates chip, is used
In the generation of microlayer model；It is microlayer model sample pipetting volume and generation microlayer model collection device with the second component, is used for first
The collection of the microlayer model of the sample-adding and generation of the oily phase sample and aqueous sample of part.
In one embodiment, the first component and the second component pass through dispensing mode or ultrasonic welding mode
It is sealedly and fixedly connected.
In one embodiment, the first component and the second component are formed by integrated injection molding respectively.
In one embodiment, the first component and the second component are thermoplastic materials, preferably poly- carbonic acid
Ester material, cyclic olefine copolymer or polymethyl methacrylate, polypropylene.
In one embodiment, the first component is provided at least one microlayer model generation unit, preferably four,
Eight and 12 microlayer model generation units；The second component is provided at least one microlayer model sample-adding and collector unit, excellent
It is selected as four, eight and 12 microlayer model generation units；The microlayer model sample-adding of each microlayer model generation unit corresponding thereto
Cooperate sample-adding, generation and the collection for carrying out microlayer model with collector unit.
In one embodiment, the microlayer model generation unit includes oily phase sample reception mouth, oily phase micro-pipe road, water phase
Sample reception mouth, water phase micro-pipe road and microlayer model outlet.
In one embodiment, the microlayer model generation unit includes two oily phase micro-pipe roads and a water phase micro-pipe
Road or an oily phase micro-pipe road and two water phase micro-pipe roads；The oil phase micro-pipe road and water phase micro-pipe road form cross and hand over
Structure is pitched, microlayer model is used to form.
In one embodiment, oily phase sample pipetting volume slot, oil mutually sample-adding through-hole, water are provided with above the second component
Phase sample pipetting volume slot and water phase are loaded through-hole, wherein mutually sample-adding through-hole and water phase sample-adding through-hole are separately positioned on institute to the oil
It states in oily phase sample pipetting volume slot and aqueous sample sample-adding trench bottom, the oil phase sample and the aqueous sample pass through respectively
They enter the first component；Sample collection device is provided with below the second component.
In one embodiment, the volume of the oily phase sample pipetting volume slot and the aqueous sample loading slot is respectively 1
~900 microlitres, preferably 5~500 microlitres, more preferably 100~200 microlitres.
In one embodiment, the sample collection device is included in the respectively arranged generation microlayer model in its both ends and receives
Mouth and microlayer model collection port, and prestore storage chamber；The microlayer model that the first component generates by the microlayer model receiving port into
Enter second component, then prestores storage chamber by described, and be finally collected from the microlayer model collection port.
In one embodiment, the microlayer model collection port is set as the outlet with oblique side wall construction, at its end
It is provided with connecting rod, microlayer model instills in microlayer model collection vessel through the connecting rod.
In one embodiment, the microlayer model collection vessel is centrifuge tube, and the connecting rod has curved wall；Institute
It states connecting rod to go deep into inside the centrifuge tube, be collected convenient for microlayer model.
In one embodiment, observation window is also set up on the second component, for cooperating optical system real-time monitoring
The microlayer model of generation.
In one embodiment, the microlayer model generating means further include third member, and the third member seals institute
The oily phase sample and aqueous sample of second component are stated, and external pressure is applied by it.
In one embodiment, it is also respectively provided on the first component and the second component convenient for the two fixation
Connect the location hole positioned.
Detailed description of the invention
It in order to more clearly explain the technical solutions in the embodiments of the present application, below will be to needed in the embodiment
Attached drawing is briefly described, it should be apparent that, the accompanying drawings in the following description is only some embodiments as described in this application, right
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings
Its attached drawing.
Fig. 1 is microlayer model generating means general structure schematic diagram of the invention；
Fig. 2 is microlayer model generating means first component structural schematic diagram of the invention；
Fig. 3 is that microlayer model generating means first component microlayer model of the invention generates " cross structure ", schematic diagram；
Fig. 4 is microlayer model generating means second component structural schematic diagram of the invention；With
Fig. 5 is the collection device structural schematic diagram of second component of the invention.
Specific embodiment
In order to make art technology field personnel more fully understand the technical solution in the application, below in conjunction with following knot
Closing embodiment, the invention will be further described, it is clear that and described embodiments are only a part of embodiments of the present application, without
It is whole embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not before making creative work
All other embodiment obtained is put, shall fall within the protection scope of the present application.It is right with reference to the accompanying drawings and embodiments
The present invention is further described.
One, microlayer model generating means structure
As shown in Figure 1, microlayer model generating means of the invention include: the first component 1 and second component 2, the first component 1 is
Microlayer model generates chip, and second component 2 is microlayer model sample pipetting volume and generation droplet collection device.The first component 1 and second
Part 2 is fixedly connected.
In some embodiments, microlayer model generating means of the invention further include third member 3, and third member is to be used for
Seal the microlayer model oil phase sample pipetting volume slot and aqueous sample loading slot of second component 2.Such as watertight potting pad of third member 3,
Such as prepared with silica gel, the watertight potting pad can on stomata can be set, for by its apply pressure, effect be guarantor
The crimping air-tightness between external pressure source and second component is demonstrate,proved, and convenient for applying external pressure.By means of external pressure, two
Kind sample is flowed into the first component from second component, into the fluid channel of the first component.Finally, the microlayer model generated is collected
Into microlayer model collection vessel, such as centrifuge tube (EP pipe).
As shown in Figures 2 and 3, in the present embodiment, 8 microlayer model generation units are arranged in parallel on the first component 1, from a left side to
The right side, on the first component 1, each microlayer model generation unit has independently produced each equidistant arranged in parallel of microlayer model generation unit
Microlayer model.The quantity of microlayer model generation unit depends on the size and life that microlayer model generates chip in the first component 1
At the needs of drop.It is to generate microlayer model " cross " structure that currently used microlayer model, which generates structure, for example, generation diameter is
100 microns of microlayer model, the width and depth of " cross " structure are 70 microns.It is viewed from above, each microlayer model generation unit
Including oily phase sample reception mouth 11, two road binders phase micro-pipe roads 12, aqueous sample receiving port 13, water phase micro-pipe road 14 and micro- all the way
Drop outlets 15.After oily phase sample enters the phase sample reception mouth 11 of the first component 1, it is diverted to two road binders phase micro-pipe roads 12.Water
After phase sample enters the aqueous sample receiving port 13 of the first component 1, into water phase micro-pipe road 14 all the way.Two-phase liquid is intersected in ten
Word structure generates microlayer model, and microlayer model is then from 15 outflow of microlayer model outlet.
Second component 2 is to realize that " oily phase " sample, the sample introduction of " water phase " sample and microlayer model are collected.As shown in figure 4,
In the present embodiment, corresponding with 8 microlayer model generation units of the first component, 8 microlayer models are arranged in parallel on second component 2
Sample-adding and collector unit, from left to right, each microlayer model is loaded and the equidistant arranged in parallel of collector unit is on second component 2,
Each microlayer model sample-adding and collector unit are matched with each microlayer model generation unit, for microlayer model generation unit water phase with
The addition of oil sample sample and the microlayer model after generation are collected.Oily phase sample pipetting volume slot 21 is provided with above second component 2, oil is added
Sample through-hole 22, aqueous sample loading slot 23 and water phase are loaded through-hole 24, wherein mutually sample-adding through-hole 22 and water phase are loaded through-hole 24 to oil
It is separately positioned on oily phase sample pipetting volume slot 21 and 23 bottom center of aqueous sample loading slot；Sample is provided with below second component 2
Collection device 25 is respectively arranged at 25 both ends of sample collection device and generates microlayer model receiving port 26 and microlayer model collection outlet
27。
In some embodiments, observation window 4 is set on second component 2, for cooperating optical system real-time monitoring to generate
Microlayer model.Observation window 4 is hollow design, and observation window 4 is located at before generating microlayer model receiving port 26, is able to observe that first
The microlayer model that part 1 generates.
As shown in figure 5, the generation microlayer model receiving port 26 of sample collection device 25 is exported with microlayer model in the first component 1
15 corresponding through-holes；It is provided with after generation microlayer model receiving port 26 and prestores storage chamber 28, the microlayer model after prestoring storage chamber 28 is collected
Outlet 27 is set as the outlet with oblique side wall construction, is provided with the connecting rod 29 for connecting with EP pipe at its end, connects
Bar 29 has curved wall.Connecting rod 29 is goed deep into inside EP pipe, collects convenient for microlayer model.
As shown in figure 5, the microlayer model generated in the first component 1 passes through microlayer model outlet 15 and second under pressure
The generation drop outlets through-hole 26 of part, into storage chamber 28 is prestored, the density of microlayer model is less than oily phase sample, can float on the upper of oil
Side, with the continuous inflow of microlayer model and oil, the oblique side wall that microlayer model can collect outlet 27 along microlayer model is slid into EP pipe.
In some embodiments, the first component 1 and second component 2 are in oily phase sample reception mouth 11 and oily mutually sample-adding through-hole
22, aqueous sample receiving port 13 and water phase sample-adding through-hole 24, microlayer model export 15 through-holes and generate the respectively surrounding of drop outlets 26
Glue of punctuating sealing.
Two, microlayer model generating means workflow
Entire workflow is divided into three steps: (1) sample feeding step, (2) microlayer model generation step, and (3) micro- liquid
Drip collection step.Firstly, " oily phase " sample and " water phase " sample are added into oily 21 He of phase sample pipetting volume slot of second component 2
Aqueous sample loading slot 23.By means of external pressure, by oil, mutually sample-adding through-hole 22 and water phase are loaded through-hole to two kinds of samples respectively
24, have respectively entered the oily phase sample reception mouth 11 and aqueous sample receiver hole 13 of the first component 1；Oil mutually enters two road binders later
Phase micro-pipe road 12, water phase enter water phase micro-pipe road 14 all the way.Oily phase and water phase are formed equal at the cross structure of the first component 1
One microlayer model.The microlayer model of generation exports the generation drop outlets 26 of 15 and second component by the microlayer model of the first component 1,
Storage chamber 28 is prestored into second component, the microlayer model for prestoring storage chamber 28 is filled by collecting outlet 27 into sample collection is connected to
The EP pipe set, completes the generation and collection of microlayer model.
(1) sample feeding step
Oily phase sample and aqueous sample are previously placed in the oily phase sample pipetting volume slot 21 and aqueous sample sample-adding of second component 2
In slot 23.External pressure effect under, two kinds of samples respectively by oil mutually sample-adding through-hole 22 and water phase be loaded through-hole 24, respectively into
Enter the oily phase sample reception mouth 11 and aqueous sample receiver hole 13 to the first component 1；Oil mutually enters two road binders phase micro-pipe roads later
12, water phase enters water phase micro-pipe road 14 all the way.
(2) microlayer model generation step
One typical " Water-In-Oil " microlayer model generating process is as shown in Figure 3: " oily phase " sample under extraneous gas pressure,
Micron level pipeline is flowed into from horizontal direction；" water phase " sample flows into micron level under extraneous gas pressure, from vertical direction
Pipeline.Two kinds of immiscible liquid cross at " cross " microfluidic structures.Due to the liquid of " oily phase " sample and " water phase " sample
Shearing force caused by body surface face tension difference and impressed pressure, " water phase " sample is at cross structure by " oily phase " sample from continuous
Mutually it is divided into discrete microlayer model.Microlayer model is the form of " Water-In-Oil ", and outside is " oily phase " sample.
(3) microlayer model collection step
The microlayer model of generation exports the generation drop outlets 26 of 15 and second component by the microlayer model of the first component 1, into
Enter second component prestores storage chamber 28, and the microlayer model for prestoring storage chamber 28 is entered by collection outlet 27 is connected to sample collection device
On EP pipe, complete the generation and collection of microlayer model.
Microlayer model flows to the microlayer model outlet of the first component 1 as seen from Figure 5 after generating in first component microchannel
15 and second component generation drop outlets 26, what drop floated to second component 2 under pressure prestores storage chamber 28, micro- liquid
The density of drop is less than oily phase sample, can float on the top of oil, and with the continuous inflow of microlayer model and oil, microlayer model can be along collection
The oblique side wall of outlet 27 slides into EP pipe, and connecting rod 29 is goed deep into inside EP pipe, collects convenient for microlayer model.
It should be understood that the present invention disclosed is not limited only to specific method, scheme and the substance of description, because these
It is alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than
It is intended to limit the scope of the invention, the scope of the present invention is limited solely by the attached claims.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein
The many equivalents for the specific embodiment of the invention stated.These equivalents are also contained in the attached claims.
Claims (15)
1. a kind of microlayer model generating means, it is characterised in that: the microlayer model generating means include the first component and second component,
The first component is fixedly connected with second component；The first component is that microlayer model generates chip, the generation for microlayer model；And institute
Stating second component is microlayer model sample pipetting volume and generation microlayer model collection device, oily phase sample and water phase sample for the first component
The collection of the microlayer model of the sample-adding and generation of product.
2. microlayer model generating means according to claim 1, it is characterised in that: the first component and the second component
It is sealedly and fixedly connected by dispensing mode or ultrasonic welding mode.
3. microlayer model generating means according to claim 1, it is characterised in that: the first component and the second component
It is formed respectively by integrated injection molding.
4. microlayer model generating means according to claim 1, it is characterised in that: the first component and the second component
It is thermoplastic material, preferably makrolon material, cyclic olefine copolymer or polymethyl methacrylate, polypropylene.
5. microlayer model generating means according to claim 1, it is characterised in that: the first component is provided at least one
Microlayer model generation unit, preferably four, eight and 12 microlayer model generation units；The second component is provided at least one
A microlayer model sample-adding and collector unit, preferably four, eight and 12 microlayer model generation units；Each microlayer model generates single
Sample-adding, generation and the collection of the microlayer model sample-adding and collector unit cooperation progress microlayer model of member corresponding thereto.
6. microlayer model generating means according to claim 5, it is characterised in that: the microlayer model generation unit includes oily phase
Sample reception mouth, oily phase micro-pipe road, aqueous sample receiving port, water phase micro-pipe road and microlayer model outlet.
7. microlayer model generating means according to claim 6, it is characterised in that: the microlayer model generation unit includes two
Oily phase micro-pipe road and a water phase micro-pipe road or an oily phase micro-pipe road and two water phase micro-pipe roads；It is described oil phase micro-pipe road and
Water phase micro-pipe road forms criss-cross construction, is used to form microlayer model.
8. microlayer model generating means according to claim 1, it is characterised in that: be provided with oily phase above the second component
Mutually sample-adding through-hole, aqueous sample loading slot and water phase are loaded through-hole for sample pipetting volume slot, oil, wherein oily mutually sample-adding through-hole and the institute
It states water phase sample-adding through-hole to be separately positioned in the oily phase sample pipetting volume slot and aqueous sample sample-adding trench bottom, the oil phase
Sample and the aqueous sample pass through them into the first component respectively；It is received with sample is provided with below the second component
Acquisition means.
9. microlayer model generating means according to claim 8, it is characterised in that: the oil phase sample pipetting volume slot and the water
The volume of phase sample pipetting volume slot is respectively 1~900 microlitre, preferably 5~500 microlitres, more preferably 100~200 microlitres.
10. microlayer model generating means according to claim 8, it is characterised in that: the sample collection device is included in it
The respectively arranged generation microlayer model receiving port in both ends and microlayer model collection port, and prestore storage chamber；What the first component generated
Microlayer model enters second component by the microlayer model receiving port, then prestores storage chamber by described, and finally from micro- liquid
Drop collection port is collected.
11. microlayer model generating means according to claim 10, it is characterised in that: the microlayer model collection port is set as having
There is the outlet of oblique side wall construction, be provided with connecting rod at its end, microlayer model instills microlayer model collection vessel through the connecting rod
In.
12. microlayer model generating means according to claim 11, it is characterised in that: the microlayer model collection vessel is centrifugation
Pipe, the connecting rod have curved wall；The connecting rod is goed deep into inside the centrifuge tube, collects convenient for microlayer model.
13. -12 any microlayer model generating means according to claim 1, it is characterised in that: also set up on the second component
Observation window, the microlayer model for cooperating optical system real-time monitoring to generate.
14. -12 any microlayer model generating means according to claim 1, it is characterised in that: the microlayer model generating means are also
Including third member, the third member seals the oily phase sample and aqueous sample of the second component, and outer by its application
Portion's pressure.
15. -12 any microlayer model generating means according to claim 1, it is characterised in that: the first component and described
It is also respectively provided on two components and is fixedly connected with the location hole positioned convenient for the two.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711074985.2A CN109746062B (en) | 2017-11-06 | 2017-11-06 | Micro-droplet generation device |
| PCT/CN2018/113851 WO2019086018A1 (en) | 2017-11-06 | 2018-11-03 | Droplet generation apparatus |
| JP2020543685A JP7030361B2 (en) | 2017-11-06 | 2018-11-03 | Microdroplet generator |
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|---|---|---|---|
| CN201711074985.2A CN109746062B (en) | 2017-11-06 | 2017-11-06 | Micro-droplet generation device |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110331088A (en) * | 2019-08-17 | 2019-10-15 | 新羿制造科技（北京）有限公司 | Microlayer model fluorescence detecting system and method |
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