CN107475101A - A kind of digital micro-droplet PCR device based on high voltage electrostatic technique - Google Patents
A kind of digital micro-droplet PCR device based on high voltage electrostatic technique Download PDFInfo
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- CN107475101A CN107475101A CN201710844092.5A CN201710844092A CN107475101A CN 107475101 A CN107475101 A CN 107475101A CN 201710844092 A CN201710844092 A CN 201710844092A CN 107475101 A CN107475101 A CN 107475101A
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- 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
- B01L3/502769—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 multiphase flow arrangements
- B01L3/502784—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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L2200/06—Fluid handling related problems
- B01L2200/0626—Fluid handling related problems using levitated droplets
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- 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
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Abstract
The present invention relates to a kind of digital micro-droplet PCR device based on high voltage electrostatic technique, mainly there are two parts, be aqueous phase droplets generation first, followed by Water-In-Oil drop is formed in fixed position.Aqueous phase droplets generating portion therein includes the needle tubing of conical nozzle, the syringe being connected with needle tubing, and the high-voltage pulse being applied on needle tubing.The described battery lead plate above microwell plate of the fixed position formation Water-In-Oil drop part including holding oil phase drop, microwell plate.By controlling the voltage between hf and hv pulse signal and battery lead plate, Water-In-Oil drop can be produced in order on microwell plate.Finally enter performing PCR amplification and detection to the microwell plate for being paved with Water-In-Oil drop, realize book digital micro-droplet PCR.The present invention is simple in construction, and consumables cost is low, and microlayer model generation and control condition are easy, can substantially reduce the experiment complexity and cost of digital pcr.
Description
Technical field
The present invention relates to a kind of digital micro-droplet PCR device based on high voltage electrostatic technique, is examined suitable for low abundance nucleic acid
Survey, can be applied to automatic biological, the field such as medical science detection, molecule diagnosis.
Background technology
The principle of droplet type digital pcr (Droplet Digital PCR) and real-time fluorescence PCR, which is distinguished, to be, Qian Zhe
Droplet processing is carried out to sample first before amplification, after PCR is expanded, the fluorescence signal of each microlayer model detected one by one
Analysis, finally according to Poisson distribution principle and the number and ratio of positive microlayer model, draw the starting copy number or dense of target molecule
Degree.Microlayer model because its form stable, be easy to manipulate and be not easy to pollute the features such as, make it the ideal chose of microreactor.
Therefore, it is stable to be quickly generated premise that a large amount of microlayer models are digital pcrs, it is necessary to the volume of caused microlayer model is homogeneous, ensure
As a result reproducible, variance is small.Meeting jet crushing is into powered droplet under electrostatic force for liquid, and charged droplet is in electrostatic field
Lower surface adsorption capacity and activity increase are acted on, causes reduction in surface tension, so as to reduce atomization resistance, changes the dynamic of drop
Can, namely change drop surface pressing it is poor.Again because the movement locus of charged drop is theoretically determined by electric-field intensity
, it is possible to apply the electrostatic fields of different voltages to control the movement locus of drop, produce the dispersion liquid of various different scales
Drop.Traditional digital pcr technology generally use dilutes by hand and distributes sample into microwell plate step by step, is carrying out follow-up amplification
And detection.This mode is cumbersome, and flux is small, and efficiency is low, and less microlayer model decomposes number and also limit its precision
With measurable dynamic range.A kind of microlayer model printing equipment involved in the present invention, the micro- of Water-In-Oil is formed using electric field action
Drop, single drop can be manipulated, simple in construction, cost low price, easily operated popularization.
The content of the invention
It is an object of the present invention to provide a kind of digital micro-droplet PCR device based on high voltage electrostatic technique, the device mainly leads to
Cross high-voltage electrostatic field and produce high speed charged drop, drop enters in oil phase pond the drop for forming Water-In-Oil, is mainly used in numeral
In droplet PCR experiment.
The technical scheme is that：
A kind of digital micro-droplet PCR device based on high voltage electrostatic technique, it is main to include the fixed bottom plate of insulation, aqueous phase droplets generation
Component, Water-In-Oil drop fixed position form component.
Described aqueous phase droplets formation component includes needle tubing, and contact tube is connected with needle tubing, the mozzle being connected with contact tube,
The syringe being connected with mozzle, and the metal base plate of electric field can be formed with needle tubing and contact tube.
Described Water-In-Oil drop fixed position forms the microwell plate that component includes holding oil phase liquid, above microwell plate
Battery lead plate.
Described needle tubing and contact tube is metal, and the wire for wherein having extraction on contact tube connects high-voltage pulse signal,
Needle tubing and contact tube are fixed on a cross fixed mount center；The metal base plate connects the ground terminal of signal.
The oil phase with surface-active is contained with the described microwell plate for forming Water-In-Oil drop component in fixed position
Liquid；Described battery lead plate is divided to two pairs, and formation is square to be distributed in above microwell plate, wherein each relative battery lead plate forms one
It is right.There is the wire of access voltage on each pair battery lead plate, two pairs of electrodes plate accesses different voltage, each pair electricity by wire respectively
Electric field is formed between pole plate, two pairs of electrodes plate forms the plane electric fields being added with after being powered above microwell plate, from the needle tubing
The charged drop drippage process ejected passes through plane electric fields, is changed by electric field force action path, is finally dropped in microwell plate
In position change.Aqueous phase droplets fall and oil phase drop are quickly injected on microwell plate because jet velocity is very fast, form Water-In-Oil
Drop.The surfactant of oil phase drop to merge between the drop to be formed.
It is fixed on the fixed bottom plate of insulation that aqueous phase droplets formation component and Water-In-Oil the drop fixed position forms component.
PCR aqueous phase reagent is preinstalled with described syringe, and is fixed in syringe fixed block.
The mozzle can be Teflon flexible pipe.
The PCR pre-installed in the syringe aqueous phase reagent is injected in the needle tubing at the appropriate speed, on needle tubing
The high-voltage pulse of application, discontinuity high-voltage electrostatic field is formed between metal base plate so that charge agent at needle tubing mouth by
The draw of electric field force and eject, be broken produce microlayer model.Pass through the controllable production of size and frequency for changing pulse voltage
The particle size of raw drop.After aqueous phase droplets eject, and influenceed by variable electric field caused by the battery lead plate above microwell plate,
Control aqueous phase droplets to be dropped in microwell plate diverse location by two pairs of electrodes plate electric-field intensity superposition, and with microwell plate oil phase
Droplet coalescence forms Water-In-Oil microlayer model.After Water-In-Oil drop is paved with the microwell plate, thermal cycle operation is carried out to it, then existed
Independently enter performing PCR amplification in each drop.
Beneficial effects of the present invention：The present invention can be achieved to produce single microlayer model, and can be to microlayer model size, drippage
Position is accurately controlled, and compared with conventional numerical PCR method, structure of the invention is simple, and consumables cost is low, microlayer model generation
And control condition is easy, the experiment complexity and cost of digital pcr can be substantially reduced.
Brief description of the drawings
Fig. 1 schematic structural views of the invention.
Fig. 2 structure top views of the present invention.
Wherein：1- needle tubings, 2- contact tubes, 3- mozzles, 4- contact tube wires, 5- syringes, 6- metal base plates, 7- electrodes
Plate wire, 8- microwell plates, 9- battery lead plates one, 10- battery lead plates two, 11- crisscross fixers, 12- aqueous phase microlayer models, 13- insulation are solid
Determine bottom plate, 14- syringe fixed blocks.
Embodiment
The present invention is done and further explained with reference to embodiment and attached Fig. 1 and 2.The following example is merely to illustrate
The present invention, but it is not used to limit the practical range of the present invention.
A kind of digital micro-droplet PCR device based on high voltage electrostatic technique, as illustrated in fig. 1 and 2, including the fixed bottom plate of insulation
13, aqueous phase droplets formation component, Water-In-Oil drop fixed position forms component.Described aqueous phase droplets formation component mainly contains
Needle tubing 1, contact tube 2, mozzle 3, contact tube wire 4, syringe 5, metal base plate 6；The Water-In-Oil drop fixed position shape
Include microwell plate 8, two teams' battery lead plate 1 and battery lead plate 2 10, battery lead plate wire 7 into component.
Before aqueous phase microlayer model 12 produces, PCR aqueous phase reagent is preinstalled with syringe 5, oil phase liquid is filled in microwell plate 8
Body, oil phase liquid are modified by surface-active so that are not merged between the Water-In-Oil drop of formation.The needle tubing 1, it is conductive
Pipe 2, mozzle 3 and syringe 5 are sequentially connected so that the aqueous phase reagent in syringe 5 can be flowed into needle tubing 1 by pipeline.
Needle tubing 1 is fixed on the center of crisscross fixer 11, and syringe 5 is fixed in syringe fixed block 14.Metal base plate 6 is fixed on
On 13 on the fixed bottom plate of insulation, microwell plate 8 is placed on metal base plate 6, and two pairs of electrodes plate besieged city is square to be placed on microwell plate 8
Side.
The reagent of PCR in syringe 5 is injected in needle tubing 1 at the appropriate speed, contact tube wire 4 connects high-tension pulse
Punching, the wired earth of metal base plate 6, discontinuity high-voltage electrostatic field is formed between such needle tubing 1 and metal base plate 6 so that needle tubing 1
The charge agent at interior place is ejected by the draw of electric field force, is broken generation aqueous phase microlayer model 12.By changing pulse
The size and frequency of voltage and the controllable particle size for producing drop of the caliber size of needle tubing 1 of customization.
After battery lead plate wire 7 on battery lead plate 1 and battery lead plate 2 10 is powered, electric field is produced in each pair battery lead plate.Cause
This aqueous phase droplets 12 is influenceed after 1 mouthful of needle tubing ejects, and by electric field caused by the two pairs of electrodes plate of the top of microwell plate 8, is changed
Become the path to fall.By controlling the power-on voltage on each pair battery lead plate wire 7, change the electric-field intensity in battery lead plate.Two pairs
Battery lead plate electric-field intensity superposition causes aqueous phase microlayer model 12 to be dropped in microwell plate diverse location, and due to the speed of whereabouts
Very fast, impulsive force is larger, with the oil phase liquid rapid fusion in microwell plate 8, forms Water-In-Oil microlayer model.When in microwell plate 8
After Water-In-Oil drop is paved with, thermal cycle operation is carried out to it, then independently enters performing PCR amplification in each drop.
Claims (4)
- A kind of 1. digital micro-droplet PCR device based on high voltage electrostatic technique, it is characterised in that：It is main to include the fixed bottom of insulation Plate, aqueous phase droplets formation component, Water-In-Oil drop fixed position form component；The aqueous phase droplets formation component includes needle tubing, Contact tube is connected with needle tubing, the mozzle being connected with contact tube, the syringe being connected with mozzle, and can be with needle tubing and conduction Pipe forms the metal base plate of electric field；The Water-In-Oil drop fixed position, which forms component, includes microwell plate, two pairs of electrodes plate.
- A kind of 2. digital micro-droplet PCR device based on high voltage electrostatic technique according to claim 1, it is characterised in that： The needle tubing is electrically conductive, there is the wire that can connect high frequency voltage on the contact tube.
- A kind of 3. digital micro-droplet PCR device based on high voltage electrostatic technique according to claim 1, it is characterised in that： The metal base plate is fixed on insulation fixed floor, and the microwell plate is on the metal base plate.
- A kind of 4. digital micro-droplet PCR device based on high voltage electrostatic technique according to claim 1, it is characterised in that： The two pairs of electrodes plate surrounds square above the microwell plate, and each pair battery lead plate has the wire of extraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710844092.5A CN107475101A (en) | 2017-09-19 | 2017-09-19 | A kind of digital micro-droplet PCR device based on high voltage electrostatic technique |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710844092.5A CN107475101A (en) | 2017-09-19 | 2017-09-19 | A kind of digital micro-droplet PCR device based on high voltage electrostatic technique |
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| Publication Number | Publication Date |
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| CN107475101A true CN107475101A (en) | 2017-12-15 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201710844092.5A Pending CN107475101A (en) | 2017-09-19 | 2017-09-19 | A kind of digital micro-droplet PCR device based on high voltage electrostatic technique |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998343A (en) * | 2018-07-25 | 2018-12-14 | 东莞东阳光科研发有限公司 | A kind of generating device of digital pcr microlayer model |
| WO2021037218A1 (en) * | 2019-08-30 | 2021-03-04 | 北京达微生物科技有限公司 | Sample adding needle for preparing microdroplets and microdroplet preparation method |
-
2017
- 2017-09-19 CN CN201710844092.5A patent/CN107475101A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998343A (en) * | 2018-07-25 | 2018-12-14 | 东莞东阳光科研发有限公司 | A kind of generating device of digital pcr microlayer model |
| WO2021037218A1 (en) * | 2019-08-30 | 2021-03-04 | 北京达微生物科技有限公司 | Sample adding needle for preparing microdroplets and microdroplet preparation method |
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