CN205501281U - Special biochip of droplet formula digit PCR - Google Patents
Special biochip of droplet formula digit PCR Download PDFInfo
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- CN205501281U CN205501281U CN201620254692.7U CN201620254692U CN205501281U CN 205501281 U CN205501281 U CN 205501281U CN 201620254692 U CN201620254692 U CN 201620254692U CN 205501281 U CN205501281 U CN 205501281U
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Abstract
The utility model discloses a special biochip of droplet formula digit PCR, including substrate, micropore array piece and cover plate, the substrate openly is equipped with and holds the recess of micropore array piece, the cover plate will with the openly cooperation of substrate the recess surrounds and forms the hermetic cavity body, grooved underside is equipped with the protruding foot of fixed stay micropore array piece, micropore on the micropore array piece is the through -hole. The utility model discloses the protruding foot of special biochip of droplet formula digit PCR through be equipped with fixed stay micropore array piece at the positive grooved underside of substrate, the micropore on the micropore array piece is the through -hole, and when using, micropore array piece is maked somebody a mere figurehead in the recess of substrate for droplet solution during the reaction is all wrapped up by the oil phase medium from top to bottom, is favorable to accurate controlled reaction's temperature.
Description
Technical field
This utility model relates to biological detection analytical equipment field, particularly relates to a kind of droplet type digital pcr special biochip.
Background technology
Droplet type digital pcr (droplet digital PCR, ddPCR) is the most emerging a kind of round pcr.On the basis of original round pcr, this technology has carried out significantly reforming.Every a example reaction system in normal PCR is divided into 20000 individual droplets by this technology, carries out PCR reaction the most again.When making microdroplet, only part microdroplet can wrap up genes of interest.PCR reacts after terminating by the fluorescence signal value of all microdroplets of equipment Inspection.Threshold value further according to fluorescence signal distinguishes the signal value of each microdroplet, is judged to that this microdroplet has wrapped up genes of interest higher than the microdroplet signal value of threshold value, is referred to as positive microdroplet.Microdroplet signal less than threshold value, it is determined that for this microdroplet no parcel to genes of interest, be referred to as negative microdroplet.Microdroplet interpretation containing fluorescence signal is 1, and the microdroplet interpretation not having fluorescence signal is 0, according to Poisson distribution principle and the number of positive microdroplet and ratio, calculates the starting copy number of target molecule.
Compared with classical quantitative fluorescent PCR, DNA or RNA molecule can be used the mode of absolute quantitation to be analyzed by ddPCR, have unrivaled accuracy, it is no longer necessary to standard curve.DdPCR provides independent non-interfering atomic little confined reaction micropore, it is possible to template molecule is really become and starts for template with single molecule, carry out the process of PCR amplification, it is possible to distinguish the extremely low content target nucleic acid molecule in sample mixing template.DdPCR has been used for the fields such as the discovery of Cancer Molecular mark, infectious disease research, genome structure analysis of variance, gene expression analysis.
CN105132571A discloses and a kind of utilizes droplet type digitized PCR detection peripheral blood to dissociate the method for mitochondrial DNA content.Described method includes that the structure of recombiant plasmid, sample are prepared, extract plasma dna and Exosome DNA, blood plasma is carried out pretreatment, and uses the steps such as droplet type digitized pcr analysis recombiant plasmid mtDNA copy number.Described method not only possesses the feature being better than the high sensitivity of qPCR, specificity and repeatability, and it is higher than qPCR to chaff interference (such as anticoagulant etc.) tolerance.Additionally, this invention is on the basis of setting up detection method, further with plasma sample directly as the template of ddPCR, carry out the analysis of blood plasma mtDNA, not only eliminate owing to extraction is brought detection uncertainty, easily facilitate Clinical Laboratory.
CN105296663A discloses a kind of based on the method for tubercule bacillus in fluorescent dye determination numeral microdroplet PCR detection blood, is, comprises the following steps: step (1) gathers blood, extracts the STb gene in blood；STb gene is carried out numeral microdroplet PCR reaction by step (2), after reaction terminates, detect the fluorescence signal value of all microdroplets and set threshold value, threshold value according to fluorescence signal determines whether microdroplet wraps up mycobacterium tuberculosis dna, mycobacterium tuberculosis dna is wrapped up higher than the microdroplet of threshold value, for positive microdroplet, do not wrap up tubercule bacillus for negative microdroplet less than the microdroplet of threshold value；The positive microdroplet number of step (3) statistics, calculates the copy number of mycobacterium tuberculosis dna, then obtains total copy number of mycobacterium tuberculosis dna in blood.The method sampling that this invention provides is convenient and swift, it is only necessary to gathers micro blood and i.e. can detect that the existence of trace tulase, and highly sensitive, detection is quickly, it is only necessary within 2 hours, just can complete detection process.
The microwell array sheet of existing droplet type digital pcr special biochip bonds with the bottom portion of groove in substrate front, micropore on microwell array sheet is not through hole, the microwell array of every chip comprises 20000 micropores, detection for a sample, each micropore can accommodate a microdroplet inside, after aqueous phase reactions liquid has configured, i.e. adds oil phase, it is initially formed Water-In-Oil structure, then into reactor carries out PCR.
Utility model content
This utility model is for the deficiencies in the prior art, it is provided that the more accurate droplet type digital pcr special biochip of a kind of temperature control.
A kind of droplet type digital pcr special biochip, including substrate, microwell array sheet and cover plate, described substrate front is provided with the groove accommodating described microwell array sheet, described cover plate coordinates the cavity that described groove surrounds formation sealing with the front of substrate, described groove floor is provided with the fixing convex foot supporting microwell array sheet, and the micropore on described microwell array sheet is through hole.
Comprising 20000 micropores on each microwell array sheet, for the detection of a sample, each micropore can accommodate a microdroplet.
Preferably, described groove floor is provided with the limit convex foot coordinated with described microwell array sheet corner.Preferably, limit convex foot is 4 right, and each angle of microwell array sheet arranges a pair limit convex foot.
Preferably, described microwell array sheet is adhesively fixed by binding agent with convex foot.Described binding agent is epoxy resin secrecy glue or silica adhesive.
Preferably, the back side of described substrate is provided with limited impression.Described limited impression is arranged at the edge of substrate back, for this utility model droplet type digital pcr special biochip is fixed to droplet type digital pcr reaction kit.
Preferably, the back side of described cover plate is provided with the boss stretching into groove.The location being provided with beneficially cover plate of described boss and fixing.
Preferably, with liquid feeding steam vent on described cover plate.After reactant liquor adds, it is initially charged part oil-phase medium, then covers cover plate, add oil-phase medium by liquid feeding steam vent, until filling it up with oil-phase medium and air being drained, after adding oil-phase medium, by liquid feeding vent hole seal.Arranging of liquid feeding steam vent conveniently drains air.Described oil-phase medium may is that alkanes, silicone oil, fluorinated oil.
Preferably, described convex foot is located at the edge of microwell array sheet, and top is provided with and the ledge structure of microwell array sheet edge mate.Described convex foot quantity is 4, and each convex foot is fixed and supported microwell array sheet, and microwell array sheet is set up on the relatively low one-level step inside described ledge structure, and the side of microwell array sheet props up the side of higher one-level step.
Preferably, described microwell array sheet surface is hydrophilic treated.The method of described hydrophilic treated is: after high-temperature hot growing surface oxide-film, be carried out with hydrophilic reagent.Described hydrophilic reagent may is that acetone, ethanol, ethylene glycol,.Preferably, described hydrophilic reagent is acetone.Microwell array sheet surface is hydrophilic treated makes water miscible microdroplet enter in micropore by hydrophilic interaction, simultaneously as droplet volume is sufficiently small, so microdroplet can hover in micropore by surface tension, without flowing out from the lower end of micropore.
Described microwell array sheet can mainly have silicon chip, glass, quartz and the high molecular polymer of polymethyl methacrylate one class by material selection.Preferably, described microwell array sheet is silicon chip.Silicon chip has low price relative to the microwell array sheet of other materials, processes the advantages such as simple.
The preparation method of described silicon chip comprises the following steps:
(1) take former of silicon, dry up after cleaning；
(2) at former dual coating photoresist of silicon, photoetching after drying, is carried out, development；
(3) if at the former double-sided deposition dried layer noble metal of silicon through photoetching；
(4) being placed in former for the silicon of depositing noble metal in corrosive liquid, reaction forms through hole；
(5) clean, dry up.
Former of described silicon is monocrystal silicon, and described monocrystal silicon is former of n-type silicon or former of p-type silicon.
Described drying temperature is 90～120 DEG C, and drying time is 30s～2min.Preferably, drying temperature is 100～110 DEG C, and drying time is 45s～90s.Most preferably, drying temperature is 105 DEG C, and drying time is 1min.
In described step (3), first one layer of silver of deposition, redeposited one layer of gold；Silver thickness is 3～10nm, and layer gold thickness is 5～20nm.Preferably, silver thickness is 3～7nm, and layer gold thickness is 7～15nm.Most preferably, silver thickness is 5nm, and layer gold thickness is 10nm.
Described corrosive liquid collocation method is: configuration concentration is the hydrofluoric acid solution of 4.0～8.0mol/L and hydrogenperoxide steam generator that concentration is 0.3～0.7mol/L, and both mix with volume ratio 10: 1 again.Preferably, described corrosive liquid collocation method is: configuration concentration is the hydrofluoric acid solution of 5.0～6.5mol/L and hydrogenperoxide steam generator that concentration is 0.4～0.55mol/L, and both mix with volume ratio 10: 1 again.Most preferably, described corrosive liquid collocation method is: configuration concentration is the hydrofluoric acid solution of 5.74mol/L and concentration is the hydrogenperoxide steam generator of 0.46mol/L, and both mix with volume ratio 10: 1 again.
On the convex foot that microwell array sheet has been fixed in the groove of substrate before using by described droplet type digital pcr special biochip.Described droplet type digital pcr special biochip using method is as follows:
(1) substrate and cover plate that are fixed with microwell array sheet are respectively placed in sample injector specify in loading slot and cover plate groove；
(2) in sample loads blade, volume required reactant liquor is added；
(3) start sample injector to sweep the reactant liquor loaded in blade painting in microwell array sheet surface；
(4) after being loaded, drip oil-phase medium with microsyringe, make oil-phase medium be surrounded by microwell array sheet；
(5) cover plate is placed on substrate, by binding agent, both is sticked together；
(6) continue to inject oil-phase medium, with the air in emptying chip by the liquid feeding steam vent on cover plate；
(7) drip appropriate fluid sealant at liquid feeding steam vent, make gelling solid with ultraviolet activation.
This utility model droplet type digital pcr special biochip is by being provided with the fixing convex foot supporting microwell array sheet in the groove floor in substrate front, micropore on microwell array sheet is through hole, in use, microwell array sheet is maked somebody a mere figurehead in the groove of substrate, microdroplet solution during reaction is the most all wrapped up by oil-phase medium, is conducive to accurately controlling the temperature of reaction.
Accompanying drawing explanation
Fig. 1 is the cover plate front view of this utility model droplet type digital pcr special biochip；
Fig. 2 is the substrate front view of this utility model droplet type digital pcr special biochip；
Fig. 3 is the substrate rearview of this utility model droplet type digital pcr special biochip；
Fig. 4 be in Fig. 2 A to sectional view；
Fig. 5 is that the microwell array sheet of this utility model droplet type digital pcr special biochip is placed in schematic diagram in wafer pockets；
Fig. 6 is the microwell array sheet of this utility model droplet type digital pcr special biochip, substrate and cover plate combination schematic diagram；
Fig. 7 is this utility model silicon chip preparation technology flow chart；
Fig. 8 is the micropore microgram of this utility model silicon chip；
Fig. 9 be in embodiment 4 this utility model droplet type digital pcr special biochip for analyzing the signal intensity scatterplot of low copy nucleic acid.
Detailed description of the invention
Embodiment 1 droplet type digital pcr special biochip
As shown in figs. 1 to 6, this utility model droplet type digital pcr special biochip includes substrate 1, the microwell array sheet 2 of band microwell array and cover plate 3.Substrate 1 front is provided with the groove 11 accommodating microwell array sheet 2, groove 11 bottom surface is provided with the fixing convex foot 12 supporting microwell array sheet 2, convex foot 12 quantity is 4, each fixing one side supporting microwell array sheet 2 of convex foot 12, convex foot 12 top is provided with and the ledge structure 13 of microwell array sheet 2 edge mate, microwell array sheet 2 is set up on the relatively low one-level step inside ledge structure 13, and the side of microwell array sheet 2 props up the side of higher one-level step.Groove 11 bottom surface is provided with the limit convex foot 13 coordinated with microwell array sheet 2 corner, and limit convex foot 13 quantity is 4 right, and each angle of microwell array sheet 2 arranges a pair limit convex foot 13.The back side of substrate 1 is provided with limited impression 14, limited impression 14 is arranged at the edge at substrate 1 back side, four angles of substrate 1 and wherein three limits are respectively arranged with a limited impression 14, and limited impression 14 is for being fixed to droplet type digital pcr reaction kit by this utility model droplet type digital pcr special biochip.
Micropore 21 on microwell array sheet 2 is through hole.Comprising 20000 micropores 21 on each microwell array sheet 2, form a microwell array, a microwell array sheet is used for the detection of a sample, and each micropore can accommodate a microdroplet inside.Microwell array sheet 2 is adhesively fixed by binding agent with convex foot 12.Microwell array sheet 2 surface is hydrophilic treated so that water miscible microdroplet enters in micropore 21 by hydrophilic interaction, simultaneously as droplet volume is sufficiently small, so microdroplet can hover in micropore 21 by surface tension, without flowing out from the lower end of micropore 21.
The back side of cover plate 3 is provided with the boss 31 stretching into groove 11.With liquid feeding steam vent 32 on cover plate 3.Cover plate 3 coordinates the cavity that groove 11 surrounds formation sealing with the front of substrate 1.
Prepared by embodiment 2 silicon chip
Silicon chip preparation technology flow process is as shown in Figure 7.Specifically comprise the following steps that
(1) by resistivity 0.1～1 Ω cmp, thickness 300 μm former of silicon ultrasonic cleaning each in acetone and ethanol 5 minutes, clean twice.Then being respectively washed 2～3 times with hot deionized water and cold deionized water, nitrogen dries up standby.
(2) former of the silicon after cleaning being dried up, two-sided resist coating, 105 DEG C are dried 1 minute, and litho machine dual surface lithography exposes 1.4 seconds, then develops 45 seconds.
(3) utilize and on former tow sides of electron beam evaporation equipment silicon after photoetching, be sequentially depositing the silver and the gold of 10nm that thickness is 5nm.
(4) preparation 5.74mol/L hydrofluoric acid solution and 0.46mol/L hydrogenperoxide steam generator, both are mixed to prepare corrosive liquid with volume ratio 10: 1.
(5) former of silicon in step (3) is placed in prepare in corrosive liquid react, after react 7.5 hours taking-up.
(6) former of the silicon taking-up that will have etched, cleans several times with deionized water, places in corrosive liquid 10 minutes, then steeps in acetone soln and takes out after 10 minutes, then nitrogen dries up after cleaning with a large amount of deionized waters, it is thus achieved that with the silicon chip of micropore.The microstructure of micropore is as shown in Figure 8.
Prepared by embodiment 3 silicon chip
(1) by resistivity 0.01～0.09 Ω cmp, thickness 300 μm former of silicon ultrasonic cleaning each in acetone and ethanol 5 minutes, clean twice.Then being respectively washed 2～3 times with hot deionized water and cold deionized water, nitrogen dries up standby.
(2) former of the silicon after cleaning being dried up, two-sided resist coating, 105 DEG C are dried 1 minute, and litho machine dual surface lithography exposes 1.4 seconds, then develops 45 seconds.
(3) utilize and on former tow sides of electron beam evaporation equipment silicon after photoetching, be sequentially depositing the silver and the gold of 10nm that thickness is 5nm.
(4) preparation 5.74mol/L hydrofluoric acid solution and 0.46mol/L hydrogenperoxide steam generator, both are mixed to prepare corrosive liquid with volume ratio 10: 1.
(5) former of silicon in step (3) is placed in prepare in corrosive liquid react, after react 7.5 hours taking-up.
(6) former of the silicon taking-up that will have etched, cleans several times with deionized water, places in corrosive liquid 10 minutes, then steeps in acetone soln and takes out after 10 minutes, then nitrogen dries up after cleaning with a large amount of deionized waters, it is thus achieved that with the silicon chip of micropore.
Embodiment 4 droplet type digital pcr special biochip is used for analyzing low copy nucleic acid
In order to verify the performance of this utility model droplet type digital pcr special biochip, using this utility model droplet type digital pcr special biochip to expand nonsmall-cell lung cancer T790M fragment with the StepOne plus qPCR of QX100dPCR, Life company of Bio-rad company, amplification uses primer and probe to be respectively as follows: the most simultaneously
Forward primer: 5 '-GCCTGCTGGGCATCTG-3 '；
Downstream primer: 5 '-TCTTTGTGTTCCCGGACATAGTC-3 '；
Fluorescent probe 1:5 '-VIC-ATGAGCTGCGTGATGAG-MGB-NFQ-3 '；
Fluorescent probe 2:5 '-FAM-ATGAGCTGCATGATGAG-MGB-NFQ-3 '.
Experimental procedure is as follows:
(1) sample DNA is extracted；
(2) the buffer mixing of sample DNA with primer, probe and premix being obtained reactant liquor, reactant liquor composition is as follows:
(3) use sample injector to be coated with by reactant liquor to sweep in the micropore of microwell array sheet；
(4) use oil-phase medium that the micropore on the microwell array sheet of biochip is carried out sealing liquid, after covering cover plate, continue to add oil-phase medium with emptying air by liquid feeding steam vent, then liquid feeding aerofluxus sky is sealed；
(5) being placed on thermal cycle heating module by biochip, arrange response procedures, carry out pcr amplification reaction, response procedures is: 50 DEG C of reactions 2min (UNG enzyme effect)；96 DEG C of denaturations 10min；98 DEG C of degeneration 30s, 63 DEG C of annealing 2min, 40 circulations；25 DEG C of preservations；
(6) signal-obtaining and analysis, calculates target DNA or the copy number concentration=-lnP/0.75nL of RNA molecule, wherein P=feminine gender micropore quantity/total effective apearture quantity.
Fluorescence signal reads and with software algorithm when analyzing is: read the digital signal that all fluorescence intensities convert, statistical analysis, deduct whole chip background noise value, obtain significant digits signal value, the threshold baseline of certain fluorescent dye delimited by mediant (1/2 maxima and minima) digital signal value of certain fluorescent dye, for FAM signal, it is determined that be positive signal value higher than the digital signal value of threshold value, it is labeled as blueness；For VIC signal, it is determined that be positive signal value higher than the digital signal value of threshold value, it is labeled as redness；When two signals are all positive, it is labeled as green；When two signals are all negative, being labeled as yellow, then, the coordinate axes figure at X-Y bidimensional regards changes all of digital signal value, carries out sorting out judgement according to the color of signal.Wherein, the signal of VIC represents wild type, and the signal of FAM represents gene mutation.
Use the result of this utility model droplet type digital pcr special biochip as shown in Figure 9.Testing positives micropore quantity as shown in table 1 for three groups, this utility model droplet type digital pcr special biochip acquired results is close with the StepOne plus qPCR acquired results of QX100dPCR and the Life company of the product B io-rad company on market.
The positive copy number results of table 1 compares.
Claims (9)
1. a droplet type digital pcr special biochip, including substrate, microwell array sheet and cover plate, described substrate front
Being provided with the groove accommodating described microwell array sheet, described cover plate coordinates the chamber that described groove surrounds formation sealing with the front of substrate
Body, it is characterised in that described groove floor is provided with the fixing convex foot supporting microwell array sheet, the micropore on described microwell array sheet
For through hole.
2. droplet type digital pcr special biochip as claimed in claim 1, it is characterised in that described groove floor sets
There is the limit convex foot coordinated with described microwell array sheet corner.
3. droplet type digital pcr special biochip as claimed in claim 1, it is characterised in that described microwell array sheet
It is adhesively fixed by binding agent with convex foot.
4. droplet type digital pcr special biochip as claimed in claim 1, it is characterised in that the back side of described substrate
It is provided with limited impression.
5. droplet type digital pcr special biochip as claimed in claim 1, it is characterised in that the back side of described cover plate
It is provided with the boss stretching into groove.
6. droplet type digital pcr special biochip as claimed in claim 1, it is characterised in that on described cover plate with
Liquid feeding steam vent.
7. droplet type digital pcr special biochip as claimed in claim 1, it is characterised in that described convex foot is located at micro-
The edge of hole array chip, top is provided with and the ledge structure of microwell array sheet edge mate.
8. droplet type digital pcr special biochip as claimed in claim 1, it is characterised in that described microwell array sheet
Surface is hydrophilic treated.
9. droplet type digital pcr special biochip as claimed in claim 1, it is characterised in that described microwell array sheet
For silicon chip.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083438A (en) * | 2017-06-12 | 2017-08-22 | 上海捷易生物科技有限公司 | Detect primer, probe and the method for EGFR gene 20 extron T790M and C797S mutation |
| WO2019144905A1 (en) * | 2018-01-24 | 2019-08-01 | 北京光阱管理咨询合伙企业（有限合伙） | Microdroplet container, method for preparing microdroplet container, microdroplet spreading method, microdroplet formation kit, temperature control device, oil phase composition for microdroplet formation and treatment method therefor |
| CN110541023A (en) * | 2018-05-28 | 2019-12-06 | 北京中科生仪科技有限公司 | digital PCR chip |
| CN113980796A (en) * | 2021-10-25 | 2022-01-28 | 苏州缔因安生物科技有限公司 | Micro-droplet bearing chip, application thereof, nucleic acid detection module and method |
| US11666900B2 (en) | 2018-01-24 | 2023-06-06 | Sniper (Suzhou) Life Technology Co. | Motion controlling mechanism, liquid discharging nozzle, microdroplet generating device and method, liquid driving mechanism and method, microdroplet generating method, and surface processing method of liquid discharging nozzle |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083438A (en) * | 2017-06-12 | 2017-08-22 | 上海捷易生物科技有限公司 | Detect primer, probe and the method for EGFR gene 20 extron T790M and C797S mutation |
| WO2019144905A1 (en) * | 2018-01-24 | 2019-08-01 | 北京光阱管理咨询合伙企业（有限合伙） | Microdroplet container, method for preparing microdroplet container, microdroplet spreading method, microdroplet formation kit, temperature control device, oil phase composition for microdroplet formation and treatment method therefor |
| US11666900B2 (en) | 2018-01-24 | 2023-06-06 | Sniper (Suzhou) Life Technology Co. | Motion controlling mechanism, liquid discharging nozzle, microdroplet generating device and method, liquid driving mechanism and method, microdroplet generating method, and surface processing method of liquid discharging nozzle |
| US11946100B2 (en) | 2018-01-24 | 2024-04-02 | Sniper (Suzhou) Life Technology Co., Ltd. | Microdroplet container and method for manufacturing the same, method for spreading microdroplets, microdroplet-generating kit, temperature-controlling device, oil phase composition for microdroplet generating and method for treating the same |
| CN110541023A (en) * | 2018-05-28 | 2019-12-06 | 北京中科生仪科技有限公司 | digital PCR chip |
| CN110541023B (en) * | 2018-05-28 | 2022-07-26 | 北京中科生仪科技有限公司 | Digital PCR chip |
| CN113980796A (en) * | 2021-10-25 | 2022-01-28 | 苏州缔因安生物科技有限公司 | Micro-droplet bearing chip, application thereof, nucleic acid detection module and method |
| CN113980796B (en) * | 2021-10-25 | 2023-12-26 | 苏州缔因安生物科技有限公司 | Micro-droplet bearing chip, application thereof, nucleic acid detection module and method |
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Address after: 310000, No. 220, North Ring Road, Xiacheng District, Zhejiang, Hangzhou Patentee after: Navigation gene technology (Hangzhou) Co., Ltd. Address before: 310000, No. 220, North Ring Road, Xiacheng District, Zhejiang, Hangzhou Patentee before: Huadong medicine (Hangzhou) gene science and Technology Co Ltd |
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