CN108148743A - A kind of drop number pcr chip and corresponding method of detection and detecting system - Google Patents
A kind of drop number pcr chip and corresponding method of detection and detecting system Download PDFInfo
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- CN108148743A CN108148743A CN201611107369.8A CN201611107369A CN108148743A CN 108148743 A CN108148743 A CN 108148743A CN 201611107369 A CN201611107369 A CN 201611107369A CN 108148743 A CN108148743 A CN 108148743A
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- pcr
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- storage tank
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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
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00279—Features relating to reactor vessels
- B01J2219/00306—Reactor vessels in a multiple arrangement
- B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
- B01J2219/00315—Microtiter plates
- B01J2219/00317—Microwell devices, i.e. having large numbers of wells
Abstract
The present invention relates to a kind of drop number pcr chips, have there are one unit or two or more same units, the unit to include：Oil phase liquid storage tank, PCR starting reaction solution liquid storage tanks and drop storage pool, the runner of runner and the connection PCR startings reaction solution liquid storage tank for connecting the oil phase liquid storage tank converges, as the drop water conservancy diversion runner for leading to the drop storage pool, wherein described drop storage pool is positioned partially at the lower section in the oil phase liquid storage tank and PCR starting reaction solution liquid storage tanks, for collecting generated drop and carrying out pcr amplification reaction.The invention further relates to corresponding method of detection and detecting system.
Description
Technical field
The present invention relates to Microfluidic droplet digital polymerase chain reaction (PCR) field, more particularly to a kind of number of drops
Word pcr chip and corresponding method of detection.In addition the invention further relates to a kind of detecting systems with the drop number pcr chip.
Background technology
Real-time fluorescence quantitative PCR (hereinafter referred to as qPCR or qRT-PCR) technology be one kind in DNA amplification reaction with fluorescence
The method that chemical substance surveys product total amount after each PCR cycle, wherein, by internal reference or outer ginseng method to the spy in sample to be tested
Determine DNA sequence dna and carry out quantitative analysis.Due to the advantages such as qPCR technologies have accuracy high, and the range of linearity is wide, therefore, extensively
Ground is for fields such as molecule diagnosis, disease research, clinical medicine.
The amplification principle of normal PCR, the Ct values of template and the starting copy number of the template are usually followed there are linear relationship,
So foundation as the original copy number of template in quantitative detection solution.Pcr amplification product is measured using TaqMan probe
During fluorescence curve, pcr amplification product is needed to reach 1011It is a copy/microlitre saturation capacity, in order to make the PCR reaction systems of 10 μ l
Reach such production concentration after 30 cycles, PCR reaction systems at least need 103A starting template amount.If by PCR's
Volume is reduced to 10 nanoliters (nl), then single template can be detected after 30 PCR cycles.
Patent document CN103451088A (a kind of microlayer model formula pcr chip and preparation method thereof) discloses a kind of using PDMS
With material of the sheet glass as micro-fluidic chip, that is, as the generation chip of microlayer model.Wherein obtained microlayer model ruler
It is very little have many advantages, such as uniformly, interface stability, PCR reaction efficiencies it is high, but due to the use of PDMS as chip material, and PDMS material
Gas permeability it is especially good, water penetration also has, thus on such chips directly carry out PCR can cause PCR reaction solution evaporate and
Cause result inaccurate.And the chip only has the function of drop formation, without the function being detected to reaction product, institute
With the function of the chip and imperfect.In addition, the chip is also provided with single-phase drop in 3 reagent inlets is molded micro- knot
Structure, structure are complex.
Invention content
The present invention is based on following principles, and nanoliter level can be grasped other very low volume fluids using microlayer model technology
Original samples reaction solution is specifically divided into picoliters to the microlayer model of nanoliter level, in this level of microlayer model most by control
More include a target DNA or RNA templates.After PCR is completed, it can be derived by the quantity for calculating positive drop
Originate total template number of target DNA or RNA in reaction solution.Microlayer model chip based on traditional single-phase microfluidic chip technology, but
Compared to single-phase microfluidic system, due to the feature of its water/oil two-phase laminated flow, the advantage is that：Consume sample and amount of reagent more
Less, mixing velocity does not easily cause cross contamination faster, or not is easily manipulated.
The present invention proposes a kind of drop number pcr chip, and there are one unit or two or more same units, units for tool
Including：Oil phase liquid storage tank, PCR starting reaction solution liquid storage tanks and drop storage pool connect the runner of oil phase liquid storage tank and connection PCR
The runner of starting reaction solution liquid storage tank converges, and becomes the drop water conservancy diversion runner for leading to drop storage pool, wherein drop storage pool portion
Divide ground or be entirely located in the lower section that reaction solution liquid storage tank is originated in oil phase liquid storage tank and PCR, for collecting generated drop simultaneously
Carry out pcr amplification reaction.
According to the present invention, the oil phase flowed out by oil phase liquid storage tank and the starting reaction solution flowed out by starting reaction solution liquid storage tank
It crosses, generates water-in-oil type drop.The drop generated imports drop storage pool through runner and carries out pcr amplification reaction herein.
According to the present invention, the unit can include two or more PCR and originate reaction solution liquid storage tank, to accommodate difference
PCR reactants.In this case, the liquid from all PCR starting reaction solution liquid storage tanks converges to form mixture first,
Then the oil phase with being flowed out by oil phase liquid storage tank crosses, and generates water-in-oil type drop.
According to the present invention, can be tiled chamber by the individual layer drop set on chip, PCR will be passed through in drop storage pool
Drop after amplified reaction introduces individual layer drop tiling chamber, and PCR amplification production is carried out to being located at the drop in individual layer drop tiling chamber
The detection of object.Therefore, each unit of chip according to the invention further includes puts down for detecting the individual layer drop of pcr amplification product
Spread chamber and waste liquid liquid storage tank, individual layer drop tiling chamber is communicated by runner with drop storage pool, waste liquid liquid storage tank by runner and
Individual layer drop tiling chamber and drop storage pool communicate.Wherein advantageously, the chip can have upper, middle and lower three layers structure,
Structure includes the oil phase liquid storage tank, PCR starting reaction solutions liquid storage tank, waste liquid liquid storage tank at the middle and upper levels；Media layer damage includes described
Runner and drop water conservancy diversion runner and individual layer drop tiling chamber；Understructure includes drop storage pool.
According to the present invention, in another form of implementation, the drop in drop storage pool is by pcr amplification reaction
Afterwards, aspirating needle can also be used, drop is sucked out from drop storage pool, the detection of pcr amplification product is carried out to drop.Therefore,
In a kind of advantageous form of implementation of the present invention, the chip can have upper, middle and lower three layers structure, wherein superstructure packet
Include the oil phase liquid storage tank, PCR starting reaction solution liquid storage tanks；Media layer damage includes the runner and drop water conservancy diversion runner；It is described
Drop storage pool runs through three layers of the upper, middle and lower, to be inserted into aspirating needle.
The function of drop formation and PCR amplification and optional PCR are integrated with according to micro-fluidic chip proposed by the present invention
Amplified production detection function, so as to can greatly improve the digital pcr detection that low copy gene is carried out using drop.In addition, it presses
According to chip proposed by the present invention without setting single-phase drop molded micro structure in each reagent inlet, structure is more simple.
In addition advantageously, used in the application according to chip of the present invention with notch, such as rice word or cross notch
Silica gel plug is sealed each liquid storage tank.It is sealed by using the silica gel plug with rice word or cross notch, so as to
Effectively avoid cross contamination.
In addition advantageously, when chip according to the invention includes individual layer drop tiling chamber, for drop to be guided to enter
Exit portion in the runner of drop storage pool is equipped with downward drainage channel, so that the fluids in drops generated in this way directly imports institute
State drop storage pool.Design in this way, it is possible to prevente effectively from the siphonage of microcavity generates, so as to prevent drop
Flow directly into individual layer drop tiling chamber.
In one embodiment, the oil phase liquid storage tank, PCR originate reaction solution liquid storage tank, individual layer drop tiling chamber, give up
Liquid liquid storage tank and the runner form semi chip, and the drop storage pool forms lower semi chip, and the upper semi chip is under
Semi chip sealing-in is an entirety.It is the oil phase liquid storage tank, PCR startings reaction solution liquid storage tank, described in another form of implementation
Runner and the drop storage pool are partially formed semi chip, and the drop storage pool lower part forms lower semi chip, institute
It is an entirety to state semi chip and lower half chip sealing.
Advantageously, the upper semi chip and lower semi chip are an entirety through thermocompression bonding sealing-in.
In addition, chip according to the invention can be by with good optical and the material system for being resistant to PCR reaction temperatures
Into.As example, the material can be makrolon (PC) plastics or cyclic olefine copolymer (COC) material.
It, can when chip according to the invention is made of makrolon (PC) plastics or cyclic olefine copolymer (COC) material
The sealing-in of chip is realized using thermocompression bonding, so as in PCR whole process all in completely enclosed environment, therefore in this way
It can effectively avoid the problem that PCR processes generate DNA aerosols, effective solution is provided for practical application.
In addition advantageously, chip according to the invention is by makrolon (PC) plastics or cyclic olefine copolymer (COC) material
Injection molding.It is using injection molding and usually disposable in the application due to chip according to the invention, because
It is very favorable in cost that this chip according to the invention is compared to the chip using PDMS material.Additionally due to using
High temperature resistant material PC or COC make chip, then without taking out generated drop, by the bellmouth of thin-walled, (drop stores
Pond) it can directly carry out PCR temperature controls reaction (heating and cooling).
As an example, diameter of the inner cavity with 0.5cm of the oil phase liquid storage tank in chip according to the invention,
The depth of 0.8cm and the wall thickness of 1.2mm；The inner cavity of the PCR startings reaction solution liquid storage tank has diameter, the 0.8cm of 0.6cm
Depth, 1.2mm wall thickness；The runner is with 200 μm of width, 50 μm of depth；The individual layer drop tiling chamber has
The width of 0.9cm, 150 μm of depth；The shape of the drop storage pool be taper, the upper loop diameter with 0.6cm,
The lower loop diameter of 0.3cm, 18 degree of taper, the internal depth of 1cm and the wall thickness of 0.5mm.
Further it is proposed that a kind of corresponding method of detection using chip according to the invention, includes the following steps：
By oil phase and PCR starting reaction solution be respectively added to oil phase liquid storage tank and PCR starting reaction solution liquid storage tank in and
Apply the silicon rubber cup with reserved venthole；
Apply air pressure in the oil phase liquid storage tank and PCR starting reaction solution liquid storage tanks respectively and continue specific time, so as to
Make to generate a large amount of small liquid of uniform size respectively from the liquid joint of oil phase liquid storage tank and PCR starting reaction solution liquid storage tanks
Drop；With
Pcr amplification reaction is carried out after the drop of the generation all imports the drop storage pool；With
After pcr amplification reaction, PCR reaction results are read.
Advantageously, this method may also comprise the following steps:：
After pcr amplification reaction, the liquid is given through the oil phase liquid storage tank and/or PCR starting reaction solution liquid storage tanks
Drip the heavy oil that storage pool injection proportion is more than drop proportion so that the drop can flow into the individual layer drop tiling chamber；
To being located at the drop in individual layer drop tiling chamber, PCR reaction results are read.
Wherein, PCR reaction rear-guard hydrodynamic drips are being realized using the method for oil phase lifting using chip according to the invention
Enter individual layer tiling intracavitary.Since the proportion of the oil is more than the proportion of drop, the oil level of drop storage pool is injected under drop,
So as to which the liquid level for making drop storage pool is raised, drop is enable to be flowed out from outlet, be laid into the drop of individual layer, it is convenient to PCR results
Record.
Herein preferably, to being located at the drop in individual layer drop tiling chamber, the fluorescence microscope with specific filter is used
Read PCR reaction results.
Advantageously, according to method proposed by the present invention, through oil phase liquid storage tank to drop after pcr amplification reaction terminates
Using 100mbars air pressures and continue 5 minutes in the step of storage pool injection oil, so as to the liquid level sustained elevation of drop storage pool,
So that realize that drop can be flowed into from outlet in individual layer drop tiling chamber.
In another advantageous forms of implementation, this method may also comprise the following steps:：After pcr amplification reaction, lead to
Aspirating needle is crossed to be drawn onto in transparent detection device the drop after reacted from the drop storage pool；To being located at the transparent detection
Drop in device reads PCR reaction results.
Wherein preferably, PCR reaction results are read using PMT photomultiplier transits tube module.
In addition, the present invention also proposes a kind of detecting system, including：
Pcr chip according to the invention；
For the circulating heater of pcr amplification reaction；With
PCR result reading devices.
In one form, PCR results reading device can include：
Laser light source is located at right over chip area and with 45 ° of incident directions；
Varifocal zoom lens and CCD camera, they are located at right over the chip area；
Band logical fluorescent optical filter is located between the Varifocal zoom lens and CCD camera.
Advantageously, excitation light source includes light emitting diode (LED) and 15 ° of lens and band logical exciting light optical filter, wherein
The bandwidth of centre wavelength and 10nm of the band logical exciting light optical filter with 473nm.Wherein, 15 ° of lens and band logical exciting light filter
Piece is respectively used to the focusing and filtering for the light that light emitting diode is sent out, and 45 ° of oblique fire angles may insure the individual layer liquid in chip
The uniform irradiation in tiled area is dripped, so as to effectively reduce exciting light scattering background, improves the sensitivity of fluoroscopic examination.
Advantageously, the bandwidth of centre wavelength and 40nm of the band logical fluorescent optical filter with 535nm.Specifically, in the application
The fluorescence of drop internal is excited and is collected by the Varifocal zoom lens of top on chip, after band logical fluorescent optical filter filtering
Fluorescence picture is acquired into CCD camera, so as to fulfill the acquisition of PCR results.
In another advantageous forms of implementation, PCR results reading device can include：Aspirating needle and PMT photomultiplier transit pipe dies
Block.
Advantageously, PCR results reading device can also include transparent detection device.
Description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.It should be evident that the accompanying drawings in the following description only describes
The part of the embodiment of the present invention.These attached drawings are not restricted for the present invention, but are served illustrative.
Wherein：
Fig. 1 schematically illustrates the front of drop number pcr chip according to the invention and corresponding sectional view；
Fig. 2 schematically illustrates 200 flow chart of corresponding method of detection according to the invention.
Fig. 3 schematically illustrates the front of another drop pcr chip according to the invention and corresponding sectional view.
Specific embodiment
Fig. 1 schematically illustrates the front of drop number pcr chip according to the invention and corresponding sectional view.In the upper of Fig. 1
Part is the front elevation of chip, and lower part is corresponding sectional view.
As shown in the chip front side figure of part on Fig. 1, the chip is identical with upper and lower two as a kind of drop pcr chip
Unit, each unit include：Oil phase liquid storage tank 1, runner 2, PCR starting reaction solutions liquid storage tank 3, drop water conservancy diversion runner 4, drop storage
Deposit pond 5, individual layer drop tiling chamber 6, support line 7 and waste liquid liquid storage tank 8.Each liquid storage is applied in addition, being further included in application
For the silica gel plug 9 of sealing on pond.Wherein, PCR originates reaction solution liquid storage tank 3, waste liquid liquid storage tank 8, runner 2 and 4, individual layer drop
The chamber 6 that tiles forms upper semi chip, and drop storage pool 5 forms lower semi chip, and upper semi chip and lower semi chip are passed through by PC plastic
Be molded and through thermocompression bonding sealing-in be an entirety.
From the point of view of the sectional view of Fig. 1 lower middle portions, which has upper, middle and lower three-decker.Superstructure is stored up including oil phase
Liquid pool 1, PCR starting reaction solutions liquid storage tank 3, waste liquid liquid storage tank 8.Media layer damage includes the runner such as runner that each liquid storage tank is connected
2 and drop water conservancy diversion runner 4 and individual layer drop tiling chamber 6.Understructure only includes drop storage pool 5.
Wherein, the inner cavity of oil phase liquid storage tank 1 has diameter, the depth of 0.8cm and the wall thickness of 1.2mm of 0.5cm；PCR
Originating the inner cavity of reaction solution liquid storage tank 3 has the wall thickness of the diameter of 0.6cm, the depth of 0.8cm, 1.2mm；Runner 2,4 has 200
μm width, 50 μm of depth；Width of the individual layer drop tiling chamber 6 with 0.9cm, 150 μm of depth；Drop storage pool 5
Shape is taper, the internal depth of upper loop diameter, the lower loop diameter of 0.3cm, 18 degree of taper, 1cm with 0.6cm and
The wall thickness of 0.5mm.
Functionally, oil phase liquid storage tank 1 and PCR starting reaction solutions liquid storage tank 3 are divided into for that will originate reaction solution
Drop reaction solution of the volume at 0.5-1 nanoliters, i.e., of oil phase and PCR starting reaction solutions liquid storage tank 3 from oil phase liquid storage tank 1
Beginning reaction solution converges, the water-in-oil type drop reaction solution of 0.5-1 nanoliters of generation；Drop storage pool 5 is used to collect drop and carry out
PCR；After the completion of PCR, individual layer drop tiling chamber 6 is adopted for receiving the drop imported from drop storage pool 5 then to realize
Related PCR is read with suitable mode as a result, for example utilizing the fluorescence microscope of specific filter.
Particularly, since drop water conservancy diversion runner 4 dedicated for guiding drop imports drop storage pool 5, can be equipped with herein to
Lower drainage channel, so that the fluids in drops generated in this way directly imports the drop storage pool.The purpose of the design is, effectively
It prevents in the more significant liquid siphon effect of narrow flow passage chamber internal ratio.
Fig. 2 schematically illustrates the flow chart of corresponding method of detection 200 according to the invention.Method 200 includes the following steps：
By oil phase and PCR starting reaction solution be respectively added to oil phase liquid storage tank and PCR starting reaction solution liquid storage tank in and
Apply the silicon rubber cup 201 with reserved venthole；
Apply air pressure in the oil phase liquid storage tank and PCR starting reaction solution liquid storage tanks respectively and continue specific time, so as to
Make to generate a large amount of droplets of uniform size respectively from the liquid joint of oil phase liquid storage tank and PCR starting reaction solution liquid storage tanks
202；With
Pcr amplification reaction 203 is carried out after the drop of the generation all imports the drop storage pool；With
After pcr amplification reaction, PCR reaction results 205 are read.
Wherein advantageously, this method may also comprise the following steps:：After pcr amplification reaction, stored up through the oil phase
Liquid pool and/or PCR starting reaction solution liquid storage tanks overweight the heavy oil of drop to drop storage pool injection proportion so that the liquid
Drop can flow into the individual layer drop tiling chamber 204；
Then to being located at the drop in individual layer drop tiling chamber, PCR reaction results 205 are read.
Wherein, PCR reaction rear-guard hydrodynamic drips are being realized using the method for oil phase lifting using chip according to the invention
Enter individual layer tiling intracavitary.Since the proportion of the oil is more than the proportion of drop, the oil level of drop storage pool is injected under drop,
So as to which the liquid level for making drop storage pool is raised, drop is enable to be flowed out from outlet, be laid into the drop of individual layer, it is convenient to PCR results
Record.
Disposably using above-mentioned according to the chip with structure as described in Figure 1 proposed by the present invention in method 200, press
It is molded according to the chip of the present invention by makrolon (PC) plastics or cyclic olefine copolymer (COC) material injection, therefore compared to adopting
It is very favorable in cost to be with the chip of PDMS material.
Specifically, in step 201,50 μ l oil are added in oil phase liquid storage tank 1 as oil phase, 25 μ l PCR of addition rise
Beginning reaction solution is in storage pool 3.Complete addition after, apply respectively on two liquid storage tanks special single-way as shown in Figure 1 into
Aerosil cap 9；
Then, in step 202, apply 100mbars to oil phase liquid storage tank 2 and PCR starting reaction solutions liquid storage tank 3 respectively
With the air pressure of 125mbars, it is a uniform in size that about 20000-30000 can be formed in the intersection of oil phase and PCR reaction solution in this way
Microlayer model, these microlayer models formed converge to the inside of drop storage pool 5 through drop water conservancy diversion runner 4.In order to preferably collect
The drop formed devises a runner drained downwards in drop outflux, and fluid can be with stream after generating drop in this way
Road directly imports drop storage pool.The time of drop formation takes around 10 minutes.
After the drop that drop formation finishes, generates all is imported in drop liquid storage tank 5, carry out in step 203
PCR.For example, PCR reactions can be carried out according to the PCR response procedures that user specifically sets.
After PCR, 204 are entered step, wherein injecting 50-80 μ l oil, the proportion of the oil in oil phase storage pool 1
More than drop, and this two-phase is mutual exclusive to the oil with drop, then applies the gas of 100mbars to oil phase storage pool 1
Pressure about 5 minutes, optionally simultaneously to the air pressure for applying 20mbars on PCR reaction solution storage pool 3.The oil stream added as a result, enters drop
It is located at bottom in storage pool 5 so as to raise liquid level so that the drop on upper strata can be flowed into individual layer drop tiling chamber 6.Due to
The chamber height that tiles in the production process of chip is 150 μm, and the liquid-drop diameter that the chip is formed under the control of above-mentioned air pressure exists
Between 100-150 μm, therefore, the drop in the chamber that tiles only can slowly flow into the drop face to form individual layer.
Then, in step 205, fluorescence imaging is carried out using particular detection system, then by relevant software to detection
As a result analyze, can finally calculate total DNA profiling number in PCR starting reaction solutions.
Wherein the particular detection system can design as follows：Other than according to pcr chip proposed by the present invention, the detection
System can also include：For the circulating heater of PCR；Laser light source is located at right over chip area and has
45 ° of incident directions；Varifocal zoom lens and CCD camera, they are located at right over the chip area；Band logical fluorescent optical filter,
It is located between the Varifocal zoom lens and CCD camera.
In this example embodiment, excitation light source includes light emitting diode (LED) and 15 ° of lens and band logical exciting light optical filter,
The bandwidth of centre wavelength and 10nm of the wherein described band logical exciting light optical filter with 473nm.In addition, the band logical fluorescence filters
The bandwidth of centre wavelength and 40nm of the piece with 535nm.
In the concrete application of the method according to the invention, after PCR reactions terminate, the light emitting diode warp in system
By 15 ° of lens and band logical exciting light optical filter, 45 ° of individual layer drop tiled areas 6 uniformly slanted in chip above chip.
15 ° of lens and band logical exciting light optical filter are respectively used to focus on and filter.Here, 45 ° of oblique fire light path can effectively reduce excitation
Light scattering background, so as to improve the sensitivity of fluoroscopic examination.Thus after the fluorescence of excitation drop internal, the varifocal mirror of top
Head can be collected, and through band logical fluorescent optical filter filtering after enter CCD camera, by CCD camera acquire fluorescence picture so as to
Obtain PCR reaction results.
Integrated chip shown in FIG. 1 drop formation, PCR amplification and pcr amplification product detection.According to proposed by the present invention
Chip can also only integrate the function of drop formation and PCR amplification, as shown in Figure 3.
Fig. 3 schematically illustrates the front of another drop number pcr chip according to the invention and corresponding sectional view.Scheming
3 upper part is the front elevation of chip, and lower part is corresponding sectional view.Wherein 1 is oil phase storage pool, and 2 be runner, and 3 be PCR
Reaction solution storage pool is originated, 4 be drop water conservancy diversion runner, and 5 be drop storage pool, and 9 be silica gel plug, and 10 be PMT photomultipliers, 11
It is transparent detection device for aspirating needle, 12.
As shown in figure 3, other than drop storage pool 5 is containing upper and lower two parts, oil phase liquid storage tank 1, PCR in chip
Corresponding construction in starting reaction solution liquid storage tank 3, the setting of runner 2 and drop water conservancy diversion runner 4 and drop storage pool 5 and Fig. 1
It sets similar.When using chip as shown in Figure 3, oily packet is being formed according to above for the method described in chip shown in Fig. 1
Water type drop and after completing PCR amplification, can drip to transparent detection dress using the liquid draw from drop storage pool 5 of aspirating needle 11
In putting 12, then (such as utilizing photomultiplier PMT) reads correlation PCR results by the way of being suitble to.Therefore, such as Fig. 3 institutes
The chip shown need not include individual layer drop tiling chamber and waste liquid liquid storage tank.
Above description to the embodiment proposed, enables professional and technical personnel in the field to realize or use the present invention.
The present invention is not limited to the specific embodiments to enumerate.For example, those of ordinary skill in the art's principle according to the present invention, root
According to the volume of Water-In-Oil drop gone for, can to parameters (such as each liquid storage tank, runner individual layer drop tiling chamber and
The size of runner, the air pressure applied etc.) it makes and correspondingly adjusting.
It should be appreciated that the feature disclosed in above example, it, can be individually other than the situation for having special instruction
Or it is used in conjunction with.A variety of modifications to these embodiments will be apparent for those skilled in the art
, the general principles defined herein can without departing from the spirit or scope of the present invention, in other embodiments
Middle realization.Therefore, invention disclosed herein is not limited to disclosed specific embodiment, but is intended to as appended
The modification within the spirit and scope of the present invention that claims are limited.
Claims (15)
1. a kind of drop number pcr chip has there are one unit or two or more same units, the unit to include：
Oil phase liquid storage tank, PCR starting reaction solution liquid storage tanks and drop storage pool, connect runner and the connection of the oil phase liquid storage tank
The runner of the PCR startings reaction solution liquid storage tank converges, and becomes the drop water conservancy diversion runner for leading to the drop storage pool, wherein institute
It states drop storage pool and is positioned partially at the lower section that reaction solution liquid storage tank is originated in the oil phase liquid storage tank and PCR, for collecting
The drop of generation simultaneously carries out pcr amplification reaction.
2. drop number pcr chip according to claim 1, which is characterized in that the chip has upper, middle and lower three-layer type
Structure, wherein superstructure include the oil phase liquid storage tank, PCR starting reaction solution liquid storage tanks；Media layer damage includes the runner
With drop water conservancy diversion runner；The drop storage pool runs through three layers of the upper, middle and lower.
3. drop number pcr chip according to claim 1 or 2, which is characterized in that the chip is by with good optical
Property and be resistant to the materials of PCR reaction temperatures and be made.
4. chip according to claim 3, which is characterized in that the chip is total to by makrolon (PC) plastics or cycloolefin
Polymers (COC) material is made.
5. chip according to claim 4, which is characterized in that the chip is total to by makrolon (PC) plastics or cycloolefin
Polymers (COC) injection molding.
6. according to the chip described in any one of claim 1-6, which is characterized in that the oil phase liquid storage tank, PCR starting reactions
Liquid liquid storage tank, the runner and the drop storage pool are partially formed semi chip, the drop storage pool lower part shape
Into lower semi chip, the upper semi chip and lower half chip sealing are an entirety.
7. chip according to claim 6, which is characterized in that the upper semi chip and lower semi chip are through thermocompression bonding sealing-in
For an entirety.
8. according to the chip described in any one of claim 1-7, which is characterized in that stored for drop to be guided to enter drop
Exit portion in the runner in pond is equipped with downward drainage channel, so that the fluids in drops generated in this way directly imports the drop storage
Deposit pond.
9. according to the chip described in any one of claim 1-7, which is characterized in that the chip, which is matched, to be useful for each storage
The silica gel plug with rice word or cross notch that liquid pool is sealed.
10. a kind of drop PCR detection system, including：
According to the pcr chip described in any one of claim 1-9；
For the circulating heater of pcr amplification reaction；With
PCR result reading devices.
11. detecting system according to claim 10, which is characterized in that the PCR results reading device includes：Aspirating needle
With PMT photomultiplier transit tube modules.
12. detecting system according to claim 11, which is characterized in that the PCR results reading device further includes transparent
Detection device.
13. a kind of drop number PCR detection method (200), includes the following steps：
Oil phase and PCR starting reaction solutions are respectively added in oil phase liquid storage tank and PCR starting reaction solution liquid storage tanks and applied
Silicon rubber cup (201) with reserved venthole；
Apply air pressure in the oil phase liquid storage tank and PCR starting reaction solution liquid storage tanks respectively and continue specific time, to make point
The liquid joint of reaction solution liquid storage tank is not originated from oil phase liquid storage tank and PCR and generate a large amount of droplets of uniform size
(202)；With
Pcr amplification reaction (203) is carried out after the drop of the generation all imports the drop storage pool；With
After pcr amplification reaction, PCR reaction results (205) are read.
14. according to the method for claim 11, which is characterized in that the method is further comprising the steps of：
After pcr amplification reaction, the drop after reacted is drawn onto by aspirating needle by transparent inspection from the drop storage pool
It surveys in device (204)；
To being located at the drop in the transparent detection device, PCR reaction results (205) are read.
15. according to the method for claim 14, which is characterized in that being located at the drop in the transparent detection device, make
PCR reaction results are read with PMT photomultiplier transits tube module.
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