CN105838801A - Emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips - Google Patents
Emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips Download PDFInfo
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- CN105838801A CN105838801A CN201610297439.4A CN201610297439A CN105838801A CN 105838801 A CN105838801 A CN 105838801A CN 201610297439 A CN201610297439 A CN 201610297439A CN 105838801 A CN105838801 A CN 105838801A
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- microballoon
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention relates to an emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips. The emulsion droplet digital PCR quantitative method comprises the steps that firstly, the microcolumn array chips are prepared; secondly, polystyrene microspheres with the surfaces modified with streptavidin are extracted, diluted and then added into turbid liquid of target magnetic beads step by step for incubation, and a microsphere and magnetic bead composite is enriched and resuspended; thirdly, a resuspended solution of the microsphere and magnetic bead composite in the second step is taken and introduced into a device to be washed, the microsphere and magnetic bead composite intercepted on the chips is counted, and the number of the target magnetic beads is obtained, so that the purpose of high-sensitivity quantification on nucleic acid is achieved. According to the emulsion droplet digital PCR quantitative method based on the microspheres and the microcolumn array chips, the chips are easy to assemble and low in cost; the target magnetic bead counting method of a BEAMing technology is more convenient and easier to implement while the advantage of high sensitivity of a BEAMing experiment is maintained, and a practical tool easy, convenient and fast to use is provided for high-sensitivity detection of nucleic acid.
Description
Technical field
The invention belongs to emulsion droplet digital pcr field, particularly to a kind of emulsion droplet digital pcr based on microballoon and micro-pillar array chip
Quantitative approach.
Background technology
PCR (PCR reaction) is the core technology of modern molecular biology experiment, its specific amplification purpose core
The method of acid fragment improves the sensitivity of nucleic acid molecules detection, plays an important role in many biological studies and medical diagnosis on disease.
Derived many nucleic acid quantitation technique according to PCR reaction amplification principle, wherein BEAMing (beads, emulsion,
Amplification, and magnetics) it is a kind of emulsion droplet digital pcr technology [D.Dressman, H. of based on Myoglobin amplification
Yan,G.Traverso,et.al.,Transforming single DNA molecules into fluorescent magnetic particles for
detection and enumeration of genetic variations[J].Proceedings of the National Academy of
Sciences,2003,100:8817-8822].Its basic procedure is the magnetic bead and single target molecules that are modified with primer to be individually wrapped
In a Water-In-Oil cavity, target molecules replicates in magnetic bead surfaces, afterwards to emulsion droplet breakdown of emulsion, collect magnetic bead and with statistics
There is the magnetic bead number of nucleic acid amplification.By amplified reaction, single target molecules can be converted into single magnetic bead surface by BEAMing
On up to ten thousand target molecules copy, be exaggerated the signal of target molecules.Owing to single target molecules is only entered a magnetic bead surfaces
Row amplification, a target magnetic bead corresponding target molecules copy, detection target magnetic bead number just it will be seen that in solution, target divides
Copy number [F.Diehl, M.Li, Y.He, et al., the BEAMing:single-molecule PCR on microparticles in of son
water-in-oil emulsions[J].Nature Methods,2006,3:551-559.].Emulsion in BEAMing experiment can be general
Expanding in logical PCR instrument, the purpose product amplified can reclaim with separating-purifying, the most flexible and side
Just.
At present, BEAMing technology has been used for quantitatively detection [X.Shi, C.Tang, the W. of the nucleic acid fragments such as sudden change and mRNA
Wang,et al.,Digital quantification of gene expression using emulsion PCR[J].Electrophoresis,
2010,31:528-534.]、[Y.Tong,W.Zhu,X.Huang,et al.,PCR amplification from single DNA
molecules on magnetic beads in emulsion:application for high-throughput screening of
transcription factor targets[J].Nucleic Acids Research,2005,33(17):150.]、[I.Tiemann-Boege,C.
Curtis,D.N.Shinde,et al.,Product length,dye choice,and detection chemistry in the bead-emulsion
amplification of millions of single DNA molecules in parallel[J].Analytical Chemistry,2009,
81 (14): 5770-5776.], the bead diameter used by BEAMing technology is generally below 3 μm, and quantity is the biggest.Right
The counting of signal magnetic bead is a committed step in BEAMing technology.Flow Cytometry is the counting that BEAMing is conventional
Method, but the instrument of costliness and reagent limit the popularization of this technology.Research is had to be dispersed in polyacrylamide gel by magnetic bead
Make magnetic bead array for artificial counting [J.Boulanger, L.Muresan, I.Tiemann-Boege, Massively parallel
haplotyping on microscopic beads for the high-throughput phase analysis of single
molecules[J].Summaries of Technical Papers of Annual Meeting Architectural Institute of Japan D
Environmental Engineering, 2012,7 (4): 292-294.], but random magnetic bead arrangement and limited field of microscope
Also counting is caused inconvenience.
Summary of the invention
The technical problem to be solved is to provide a kind of emulsion droplet digital pcr quantitative square based on microballoon and micro-pillar array chip
Method, the method chip assembles simple, low cost；Make while remaining BEAMing experiment high sensitivity feature
The target magnetic bead method of counting of BEAMing technology is more convenient and easy, provides a simplicity, fast for the highly sensitive detection of nucleic acid
The utility of speed.
A kind of based on microballoon and micro-pillar array chip the emulsion droplet digital pcr quantitative approach of the present invention, including:
(1) will after Wafer Cleaning, gluing use deep reaction ion etching on silicon chip, etch micro-pillar array, between micro-pillar array between
Reduce away from line number, use removing of photoresist by plasma technique to obtain silicon chip mould after removing cull；By silicon chip mould silanization, will preparation
Good PDMS is cast on silicon chip mould, and PDMS peels off after being heating and curing and i.e. obtains micro-pillar array chip；
(2) polystyrene microsphere of Streptavidin is modified on extraction surface, biotin during substep joins BEAMing experiment after dilution
The suspension of the target magnetic bead modified is hatched, is enriched with microballoon bead complexes, resuspended；
(3) take the re-suspension liquid of microballoon bead complexes in step (2), be passed in micro-pillar array chip flushing, by statistics core
The microballoon bead complexes retained on sheet, obtains the number of target magnetic bead.
The H cleaned as using volume ratio 10:1 in described step (1)2SO4/H2O2Cleaning solution cleans.
In described step (1), the spacing excursion between micro-well array is 50～6 μm.
In described step (2), the diameter of polystyrene microsphere is bigger than micro-pillar array minimum spacing, and the diameter of target magnetic bead is than microtrabeculae battle array
Row minimum spacing is little.
In described step (2) resuspended for use PBST solution resuspended.
The PBS solution consisting of pH 7.4 of described PBST solution and 0.01%Tween 20.
Flushing in described step (3) is rinsed for using PBST solution.
Present invention comprises by micro-pillar array chip design preparation and surface modify Streptavidin polystyrene microsphere separation and
Counting.The target magnetic of biotin modification in the polystyrene microsphere capture BEAMing experiment of Streptavidin is modified by surface
Pearl, utilizes magnetic field by the enrichment of microballoon bead complexes and to separate with free microballoon, then utilizes micro-pillar array chip to intercept microballoon magnetic
Pearl compound, can reach to add up the purpose of magnetic bead quantity easily.Micro-pillar array chip uses based on chi between Microsphere Size and microtrabeculae
The interception principle of very little difference, and the internal structure of rule reduces the difficulty of later stage software statistics microballoon quantity.Streptavidin
(Streptavidin, SA) modifies microballoon and has preferable dispersiveness in the solution, can effectively combine one end with biotin
Target magnetic bead, forms compound.Magnet can efficiently separate magnetic bead microsphere compound and free microballoon.The hole of micro-pillar array chip
Footpath can intercept microballoon and filter Unwanted magnetic beads, and the most regular is arranged with follow-up statistics.Present invention reduces BEAMing
The cost of detection counting, improves detection efficiency.
Beneficial effect
(1) present invention counts simply, and microballoon is fully combined with magnetic bead, and each microballoon has relatively-stationary magnetic bead adsorbance, as long as
Statistics microballoon number just may know that the approximate range of magnetic bead quantity in whole solution；The regular array of micro-pillar array reduces statistics microballoon
Difficulty, it is simple to Future Software automated enumeration；
(2) BEAMing has the feature of emulsion droplet PCR multiple reaction cavity, can easily reach the reaction of hundreds of thousands level even million grades
Chamber number, does not the most increase experimental implementation difficulty simultaneously；
(3) according to testing requirement and the difference of flux, it is also possible to increase microtrabeculae number and chip area, change microsphere diameter size parameter and
Microtrabeculae spacing etc., and microballoon can be carried out different base group modification, accomplish Multiple detection；
(4) testing cost of the present invention is low, and preparation manipulation is simple, is with a wide range of applications.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of micro-pillar array chip internal of the present invention；
Fig. 2 is the schematic diagram that micro-pillar array chip of the present invention intercepts microballoon；
Fig. 3 is that micro emulsion drips and magnetic bead distribution schematic diagram；
Fig. 4 is microballoon form in supernatant and precipitation；Wherein, left figure is the microballoon in supernatant, and right figure is magnetic bead microsphere compound；
Fig. 5 is that the chip of different templates strength solution sample intercepts result.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and not
For limiting the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1
1.1 BEAMing experiments
The primer sequence of the present embodiment is as shown in table 1.5 ' ends of primer 1, with double biotin base group modifications, can stably be tied
It is combined in magnetic bead surfaces and will not depart from subsequent thermal circular response, SA is modified the magnetic bead Dynabeads M-270 (U.S.
Invitrogen company) with primer 1 in combining buffer solution (5mM pH 7.5Tris-HCl, 0.5mM EDTA, 1M NaCl)
Incubated at room 15min, the primer 1 of excess can ensure that magnetic bead surfaces SA site is saturated.Utilize magnetic field enrichment magnetic bead afterwards also
Clean three times by re-suspension liquid (20mM pH 8.4Tris-HCl, 50mM KCl), finally resuspended by re-suspension liquid.Reaction oil breast mixes
Close liquid by 7% (wt/vol) ABIL WE09 (Evonik Degussa company of Germany), 20% (vol/vol) mineral oil (U.S.
Sigma company) and 73% (vol/vol) Tegosoft DEC (Evonik Degussa company of Germany) composition.Reaction premixed liquid
Composition (40 μ L system) is as shown in table 2.A 5mm steel ball, oil breast mixing it is sequentially added in a 1.5mL centrifuge tube
Liquid and reaction premixed liquid, cover tightly lid and put in Tissuelyser-24 full-automatic sample beveller (Shanghai Jing Xin scientific & technical corporation) and shake
Swing formation Water-In-Oil drop system.Amplified reaction is carried out in standard PCR instrument (Takara company of Japan), and reaction condition includes:
94 DEG C of 2min thermal starting kinases activity, 98 DEG C of 15s sex change, 60 DEG C of 45s annealing, 72 DEG C of 75s extend, and totally 45 from sex change
DNA cloning is carried out to extending circulation.After having expanded, take out PCR reaction tube, in collection emulsion to 1.5mL centrifuge tube,
Add breakdown of emulsion buffer solution (10mM Tris-HCl (pH 7.5), 1%Triton-X 100,1%SDS, 100mM NaCl, 1mM
EDTA), fully put into 3200g in centrifuge (Eppendof company of Germany) after vibration mixing and be centrifuged 3min, suck upper strata
Above step is repeated once after oil phase.Enrichment magnetic bead, adds after removing liquid in pipe that to combine buffer solution resuspended.So far with mesh
The magnetic bead of mark copy nucleic acid information is prepared complete.
Table 1 primer sequence
| Primer | Sequence |
| Primer 1 | 5’-dualbiotin-(PEG18-spacer)-t-gagtttctcttagtgtgtgtgagtatgtgacggag |
| Primer 2 | 5’-biotin-cacacgccgacttcaggtt |
| Primer 3 | 5’-agtgtgtgtgagtatgtgacgg |
Table 2 reacts premixed liquid composition
1.2 micro-pillar array chip manufacturings
Micro-pillar array chip manufacturing includes two parts: making and the making of PDMS mould of silicon chip mould.
1.2.1 the making of silicon chip mould
Silicon chip is placed in the cleaning solution (H boiled2SO4:H2O2=10:1) in constantly rock immersion 10min.Take out silicon chip, put
Enter deionized water clean in chamber cleaning showers and dry.The rotation of positive for LC100A glue is evenly coated on the silicon chip after cleaning.After being painted with
Front baking, removes the moisture in photoresist, increases the adhesiveness of glue and silicon chip surface, then utilizes exposure imaging technique, by mask
Pattern in version is transferred on photoresist, exposes the part being etched on silicon chip.Utilize deep reaction ion etching (DRIE) at silicon
Etching a series of micro-pillar array on sheet, the spacing between microtrabeculae constantly reduces along with line number, and excursion is 50～6 μm,
Finally remove cull by removing of photoresist by plasma technique.So far, silicon chip Mold Making completes.
1.2.2 the making of PDMS mould
To making silicon chip mould silanization, then take PDMS performed polymer and curing agent by the proportions PDMS of 10:1.Will
The PDMS prepared is cast on silicon chip mould, stands 1～2h, 90 DEG C of heating 1h.Peel off after PDMS is fully cured,
Micro-well is array switching for micro-pillar array, then use blue film cladding, manual cutting and boring.Finally by slide and chip by wait from
Daughter is bonded.So far chip is completed.
1.3 Surfaces of Polystyrene Microparticles modify Streptavidin (commercially available)
1.4 testing process
Extraction SA modifies microballoon, with combining buffer solution dilution, is added step-wise in five times in the magnetic bead suspension prepared fully blow
Beating, be placed in isothermal vibration instrument and hatch 30min, 6000rpm is centrifuged 5min subsequently, sucks supernatant.Add PBST solution
(pH 7.4PBS solution, 0.01%Tween 20) is resuspended, piping and druming mixing.Enrichment magnetic bead, sucks supernatant.Use PBST
Solution is resuspended.
Micro-pillar array chip sample introduction process takes Ngatively pressurized sampling, utilizes sample introduction instrument to be passed in device by PBST solution, with 10mL/h
Speed flusher.Take microballoon and magnetic bead PBST re-suspension liquid, resuspended with PBST solution, it is passed in device.Mixed liquor
After sample introduction completes, with PBST flusher twice.In order to prevent sample introduction process gas to be mixed into, mixed liquor is caused to divide in device
Cloth is uneven, and the liquid selective of each step adds when back is near completion.Last flushing can first be put into basis of microscopic observation,
If there being magnetic bead to remain, then can increase washing time.
2 discussion of results
2.1 Cleaning Principle
This method selects the surface of a diameter of 2.8 μm to be modified with the Dynabeads M-270 magnetic bead solid phase as amplified reaction of SA
Carrier, and the saturated combination of primer 1 of double biotin groups with the band of excess.Owing to the quantity of magnetic bead is far longer than nucleic acid to be detected
The quantity of molecule, can be easy to make single magnetic bead and target molecules co-exist in a micro emulsion drip chamber according to Poisson distribution and carry out
Amplified reaction.The primer 2 of the band biotin in premixed liquid participates in the amplification of target molecules to be made to bring biotin on target magnetic bead, draws
The existence of thing 3 improves the amplification efficiency of magnetic bead surfaces target molecule.SA modifies polystyrene microsphere can capture target effectively
Magnetic bead, is slowly added to microballoon and microballoon can be made fully to be combined with target magnetic bead.Therefore magnetic bead microsphere compound can exist together along with magnetic bead
Being enriched with under magnetic field, free microballoon is then absorbed together along with supernatant.Microsphere diameter is 10 μm, and the micro-pillar array spacing of design is minimum
It is 6 μm.Microballoon can be trapped within passage (Fig. 2) because size is big with the compound of amplification magnetic bead, and free magnetic bead then can be with
Liquid and flow out chip.By the microballoon being retained down on statistics chip, it is possible to know target magnetic bead substantially number in solution.
The heat endurance of 2.2 emulsions and reagent proportioning
Emulsion heat endurance is the factor that PCR amplification experiment is critically important, and draws 0.5 μ L after thermal cycle before thermal cycle respectively
Emulsion is laid in slide, and inverted microscope (OLYMPUS company of Japan) 400 × multiple is observed, as it is shown on figure 3, newborn
The state before and after thermal cycle that drops in does not changes, and it is dispersed and has good uniformity, and illustrates that this emulsion has good heat steady
Qualitative.
2.3 quantitative result analyses
Experiment uses substep repeatedly to add the method for microballoon to ensure that SA microballoon can fully be combined with target magnetic bead, the most often
The microballoon quantity that one step adds is about at about 500, and its quantity specifically added also can adjust according to actual conditions.Fully incubate
After educating, utilizing magnet to assemble magnetic bead, Aspirate supernatant is placed on basis of microscopic observation, the such as Fig. 4 of the microballoon in supernatant left figure institute
Show, illustrate that the microballoon total amount added captures magnetic bead the most completely.With magnetic bead microsphere compound due to magnetic field richness shown in the right figure of Fig. 4
Collection effect is gathered at the bottom of pipe, and free microballoon then separates together with supernatant.
In order to verify the feasibility of the method, have chosen cDNA fragment that a segment length is 66bp as examination target.To contain
These three solution sample is detected by the method respectively by the cDNA solution of this sequence as after twice gradient dilution, and does
Blank experiment, Fig. 5 is to intercept part microballoon in chip to intercept result.Three chips are divided into and are not truncated to 287, and 148
Individual and 85 microballoons, are converted into nucleic acid copies and are about 5740,2960 and 1700 copies, and blank is tested without microballoon
Intercept and capture.This result and extension rate contrast and present preferable linear relationship, show that this method has feasibility.
Claims (7)
1. an emulsion droplet digital pcr quantitative approach based on microballoon and micro-pillar array chip, including:
(1) will after Wafer Cleaning, gluing use deep reaction ion etching on silicon chip, etch micro-pillar array, between micro-pillar array between
Reduce away from line number, use removing of photoresist by plasma technique to obtain silicon chip mould after removing cull；By silicon chip mould silanization, will preparation
Good PDMS is cast on silicon chip mould, and PDMS peels off after being heating and curing and i.e. obtains micro-pillar array chip；
(2) polystyrene microsphere of Streptavidin is modified on extraction surface, biotin during substep joins BEAMing experiment after dilution
The suspension of the target magnetic bead modified is hatched, is enriched with microballoon bead complexes, resuspended；
(3) take the re-suspension liquid of microballoon bead complexes in step (2), be passed in micro-pillar array chip flushing, by statistics core
The microballoon bead complexes retained on sheet, obtains the number of target magnetic bead.
A kind of emulsion droplet digital pcr quantitative approach based on microballoon and micro-pillar array chip the most according to claim 1, its feature exists
In: the H cleaned as using volume ratio 10:1 in described step (1)2SO4/H2O2Cleaning solution cleans.
A kind of emulsion droplet digital pcr quantitative approach based on microballoon and micro-pillar array chip the most according to claim 1, its feature exists
In: in described step (1), the spacing excursion between micro-well array is 50～6 μm.
A kind of emulsion droplet digital pcr quantitative approach based on microballoon and micro-pillar array chip the most according to claim 1, its feature exists
In: in described step (2), the diameter of polystyrene microsphere is bigger than micro-pillar array minimum spacing, and the diameter of target magnetic bead is than microtrabeculae battle array
Row minimum spacing is little.
A kind of emulsion droplet digital pcr quantitative approach based on microballoon and micro-pillar array chip the most according to claim 1, its feature exists
In: in described step (2) resuspended for use PBST solution resuspended.
A kind of emulsion droplet digital pcr quantitative approach based on microballoon and micro-pillar array chip the most according to claim 5, its feature exists
In: the PBS solution consisting of pH 7.4 of described PBST solution and 0.01%Tween 20.
A kind of emulsion droplet digital pcr quantitative approach based on microballoon and micro-pillar array chip the most according to claim 1, its feature exists
In: the flushing in described step (3) is rinsed for using PBST solution.
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| CN112858670A (en) * | 2021-01-26 | 2021-05-28 | 深圳市科瑞达生物技术有限公司 | Multiple digital ELISA detection method and microfluidic chip |
| CN113649096A (en) * | 2021-09-16 | 2021-11-16 | 苏州集微光电有限公司 | Exosome separation micro-fluidic chip and preparation method thereof |
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