CN103343092B - Method for manufacturing digital PCR (polymerase chain reaction) chip based on mineral-oil saturated PDMS (polydimethylsiloxane) material - Google Patents
Method for manufacturing digital PCR (polymerase chain reaction) chip based on mineral-oil saturated PDMS (polydimethylsiloxane) material Download PDFInfo
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Abstract
The invention relates to a method for manufacturing a digital PCR (polymerase chain reaction) chip based on a mineral-oil saturated PDMS (polydimethylsiloxane) material. The method is characterized in that the digital PCR chip based on PDMS is prepared from a PDMS monomer of a certain amount of mineral oil (liquid paraffin) and comprises an emulsion droplet generation structure and an emulsion droplet collection structure. After the emulsion droplets are made and collected on the same chip, the emulsion droplets are subjected to PCR amplification on the same chip. The phagocytosis to the oil phase in the digital PCR system by the PDMS of the chip can be avoided, the emulsion droplets can be kept stable during PCR, and the stability of the PCR can be guaranteed. In addition, compared with the existing technology of the digital PCR chip, the method provided by the invention is low in cost, is convenient to operate and has a very wide application prospect.
Description
Technical field
The present invention relates to a kind of making method of digital pcr (polymerase chain reaction) chip based on the saturated PDMS material of mineral oil, the chip of making.For low abundance detection of nucleic acids, can be applied to the fields such as biology, medical science and environmental science.
Background technology
Quantitative fluorescent PCR (Fluorescence Quantitative Polymerase Chain Reaction, FQ-PCR, qPCR) has developed into a crucial routine techniques of biology field, has greatly promoted the development of life science every field.But, the factor that affects its amplification efficiency in pcr amplification process is a lot, be difficult to ensure that the amplification efficiency between actual sample and standard model and different sample is identical, it is not invariable causing thus basis-cycle threshold (Cycling Threshold Value, Ct) that its quantitative analysis relies on.Therefore qPCR's is quantitatively " relative quantification ", and its accuracy and circulation ratio still can not meet the requirement of molecular biology quantitative analysis.In addition, due to the restraining effect of pcr amplification product to enzymic catalytic reaction, the genovariation detection method of PCR-based technology is usually helpless to low-abundance genovariation in somatocyte at present.
Digital pcr (Digital PCR, dPCR) is a kind of nucleic acid quantification method of counting based on single-molecule PCR method, is a kind of method of absolute quantitation.The main micro-fluidic or droplet method that adopts the popular research field of present analysis chemistry, is dispersed to the nucleic acid solution after Macrodilution in the microreactor or droplet of chip, and the nucleic acid-templated number of each reactor is less than or equals 1.Through after PCR circulation, there is the reactor of a nucleic acid molecule template will provide fluorescent signal like this, do not have the reactor of template just there is no fluorescent signal.According to the volume of relative proportion and reactor, just can extrapolate the nucleic acid concentration of original solution.Different from traditional quantitative PCR, digital pcr, by the method for direct census, can be realized the absolute quantitation of initiate dna template.
In addition, a kind of digital pcr or method that can identify micro-mutant in a large amount of wild-type DNA backgrounds.Because digital pcr technology can be separated template DNA molecule separately and increase in advance, this has just been avoided high abundance allelotrope nucleic acid to suppress the amplification of variant nucleic acid, has therefore improved the detector efficiency of micro-variant nucleic acid.Emulsion droplet digital pcr technology can detect the sudden change fragment that is low to moderate 0.001%, and order-checking and conventional real-time fluorescence quantitative PCR method are helpless to being less than 1% sudden change, and the detection sensitivity of therefore suddenling change can improve 1000 times.
The general operation flow process more complicated of traditional digital pcr technology, normally first by manual stepwise dilution and distribute sample to microwell plate, then microwell plate is placed in to the enterprising performing PCR reaction of thermal cycler, after finishing, reaction reads the fluorescent signal in each micropore by instrument, final according to the ratio of Poisson's distribution principle and positive droplet, binding analysis software can calculate the concentration or the copy number that provide target molecule to be checked.This mode complex operation, flux is little, efficiency is low, and less drop decomposes the dynamicrange that number has also limited its precision and can survey, and application aspect has great limitation.In recent years, new vitality has been injected in the development that develops into digital pcr technology of microflow control technique.Because microflow control technique is in the advantage aspect microfluid manipulation, make us decomposed sample can be risen to even skin upgrading for receiving, obtain more decomposed sample number, thereby greatly improve detection sensitivity, confidence level and the dynamicrange degree of digital pcr technology.In addition, microflow control technique automatization, the advantage that easy of integration, flux is high, also can greatly improve the detection efficiency of digital pcr technology.
By microflow control technique, in the advantage aspect microfluid manipulation, in recent international, existing multiple research groups and company have developed the digital pcr system based on microflow control technique.More typically comprise the BioMark based on microchamber type that Fluidigm company releases
tMthe QX100TM system based on drop type that system and Bio-Rad company release.With respect to traditional digital pcr technology, these two systems are in sensitivity, the accuracy of automatization, the detection of operation and can survey concentration dynamicrange and be all greatly improved, but still there is in actual applications certain defect, it is main because these two systems all need extra function unit to realize the decomposition of sample, cause the complexity of system higher, thereby increase the cost of system, limited its application.
PDMS(polydimethylsiloxane, polydimethylsiloxane) be that one has elastic high molecular polymer, because of its cost low, use simple, and there is good optical characteristics, good insulativity, the features such as good unreactiveness and good ventilation property, become a kind of polymer materials that is widely used in the field such as micro-fluidic.
In digital pcr field, people adopt PDMS to make emulsion droplet generating chip more, directly less at the example of PDMS chip enterprising line number word pcr amplification and analysis, major cause is that to carry out ventilation property strong due to one, two because it has good lipophilicity, the receptivity superpower to having of oiliness molecule in PCR Thermal Cycling, therefore cannot realize amplification.
Also had in recent years minority on PDMS chip, directly to carry out the example of digital pcr amplification, they avoid the mode of oil, liquid volatilization to be mainly: on 1:PDMS chip emulsion droplet dot matrix, put and stick sheet glass; 2: in chip, spraying is to polyxylene.
First kind of way recited above cannot stop that PDMS chip material itself is to mineral pick up the oil, and PDMS itself is very strong to the receptivity of mineral oil at all; Described second way processing condition more complicated, common laboratory cannot meet this condition.
In sum, the present invention intends at open a kind of digital pcr chip preparation method based on the saturated PDMS material of mineral oil.The present invention has overcome the emulsion droplet wild effect causing by force due to PDMS oil receptivity, and compared with current existing digital pcr chip technology, has cost low, advantage easy and simple to handle, and application prospect is very extensive.
Summary of the invention
The object of the invention is to set up a kind of making method of the digital pcr chip based on the saturated PDMS material of mineral oil, to reduce digital pcr cost, the step that simplifies the operation, is beneficial to promoting the use of this technology.
Making method of the present invention comprises the preparation of chip, generation and the pcr amplification of emulsion droplet.Material prepared by described chip is glass (general slide glass or cover glass), PDMS and mineral oil (as whiteruss etc.).Concrete gordian technique is as follows:
1, the preparation of chip
Due to strict Thermal Cycling of PCR reaction needed, therefore the feature of the good permeability of PDMS own becomes the huge obstacle of pcr amplification on the contrary; In addition, the counting principle of emulsion droplet PCR is to be based upon oil phase to the cutting apart of PCR reaction solution (water), and makes PCR reaction solution in the middle of oil phase, automatically be dispersed into multiple thousands of PCR reaction members independently, independently carries out separately on the basis of PCR reaction.Therefore, PDMS forms tremendous influence to the sorption of oil phase by the stability to each PCR reaction member.The preparation of chip of the present invention comprises design and the preparation of mould, first make microchannel and sample introduction groove, then be the preparation of PDMS material, the preparation of PDMS material is first to use (being whiteruss containing the mineral oil described in 100-400 μ l mineral oil in every 1ml PDMS) in the PDMS monomer of a certain amount of mineral oil, in PDMS chip crosslinking curing process, mineral oil is filled space wherein automatically, therefore can suppress to greatest extent PDMS chip the engulfing oil phase in digital pcr system of making, maintain the stability of emulsion droplet and PCR reaction in PCR reaction process.
2, the generation of emulsion droplet
The formation of micro-fluidic chip emulsion droplet can be passed through " ten " font and two kinds of chip structure forms of " T " font.By adjusting oil phase and the size of water sample channel, the difference of length, can regulate and control the situation such as size, concentration of formed emulsion droplet.Find through overtesting, inflow pipeline and sample channel height 20-120 μ m, width 20-150 μ m all can form good emulsion droplet, under the folder stream effect of paraffin oil, the skin upgrading that cutting forms or the emulsion droplet diameter dimension of upgrading of receiving all can keep stable below 150 μ m, and emulsion droplet is made, collected on same chip; Then in the enterprising performing PCR amplification of same chip.
3, pcr amplification
Chip complete sample introduction is lain on the PCR instrument with original position PCR function and is increased, the denaturation temperature of amplification and renaturation, amplification temperature all with emulsion droplet in the condition of the PCR mixed solution that wraps up identical, but soaking time proper extension.
Making method of the present invention and conventional digital pcr method ratio, consumables cost is low, and emulsion droplet formation condition and result observational technique are easy, and required testing installation common laboratory can meet.
In a word, the invention provides a kind of method of the digital pcr chip based on the saturated PDMS material of paraffin oil.It is characterized in that the PDMS monomer of a certain amount of mineral oil (whiteruss) to prepare PDMS digital pcr chip, chip comprises emulsion droplet generating structure, emulsion droplet collection structure two portions.Emulsion droplet is made, is collected on same chip, then in the enterprising performing PCR amplification of same chip.This chip has avoided PDMS to the engulfing of oil phase in digital pcr system, and is conducive to maintain the stability of emulsion droplet and PCR reaction in PCR reaction process.The present invention has following advance:
[1] the present invention has overcome the emulsion droplet wild effect causing by force due to PDMS oil receptivity;
The PDMS chip material of ordinary numbers pcr chip can be engulfed emulsion droplet and form necessary oil phase in Thermal Cycling, causes the emulsion droplet in chip unstable, even causes aqueous phase liquid to volatilize and increases unsuccessfully.The present invention fills the hole in PDMS with paraffin oil, thereby has suppressed its phagolysis to oil phase, has safeguarded the stability of emulsion droplet in pcr amplification process, for the success of pcr amplification provides guarantee.
[2] oily saturated PDMS changes PDMS surface properties, can reduce the adsorptive power of chip to enzyme, nucleic acid, fluorescence dye etc., the trouble of having avoided chip surface treatment step to bring;
Due to the high water transport property of PDMS material surface and the strong adsorptivity to apolar substance, in biological respinse, must carry out finishing to it.Paraffin oil and biomolecules have good consistency, and are difficult in absorption biological respinse as albumen, nucleic acid etc., and the biomacromolecule of fluorescence dye is therefore applied very extensive in biological respinse.The present invention fills the hole in PDMS with paraffin oil, can reach the effect of PDMS being carried out to surface modification simultaneously, and the saturated PDMS material of paraffin oil does not adsorb substantially to enzyme, nucleic acid, fluorescence dye etc., has further ensured the stability of experiment.
[3] digital pcr method involved in the present invention is compared with current digital pcr chip technology, and cost is low, easy and simple to handle, and routine test equipment can meet test needs.
At present commercially available digital pcr chip adopts special plant and instrument to generate emulsion droplet and read signal, and whole plant is expensive, and non-common laboratory can be born.Digital pcr chip manufacture method involved in the present invention, adopts easy negative pressure pump (being worth hundreds of unit) can realize emulsion droplet and makes, and data read and adopt general fluorescent microscope to satisfy the demand, and chip material employing PDMS, and cost is very low.And whole emulsion droplet generation, collection and pcr amplification process all carry out on same chip, therefore the method cost is low, simple and easy to do, is easy to promote.
Brief description of the drawings
Fig. 1: oily saturated PDMS digital pcr floor layout;
A: chip structure; B: the cross decussate texture partial enlarged drawing of emulsion droplet generating portion; C: emulsion droplet collecting zone border microcell dam structure partial enlarged view;
Fig. 2: oily saturated PDMS digital pcr chip detection principle and step schematic diagram;
A: emulsion droplet generating principle schematic diagram; B:PCR amplification is analyzed schematic diagram;
Fig. 3: oily saturated PDMS digital pcr chip detection result;
Digital pcr probe is that FAM modifies, and white emulsion droplet is the positive emulsion droplet of amplification, and ash, black emulsion droplet are the negative emulsion droplet of amplification.
Embodiment
Explain outstanding feature of the present invention and marked improvement by specific embodiment below, but the present invention is only confined to by no means embodiment.
Embodiment 1: the designing and making (Fig. 1) of mould
The present invention has studied plurality of chip structures and positive/negative-pressure sample introduction emulsion droplet has been formed the impact of stability, finally find cross clamp stream (i.e. " ten " font) Ngatively pressurized sampling mode to external equipment require lowly, generation emulsion droplet dimensional stability is better.
Described chip is collected two portions by emulsion droplet generation, emulsion droplet and is formed, and adopts cross clamp stream negative pressure mode to generate emulsion droplet.First utilize CAD software design pattern to print mask, then utilize photoresist material SU8 2050 on silicon chip, to make microchannel and sample introduction groove structure (see figure 1), oil phase sample channel width: 200 microns, PCR mixed solution sample channel: 60 microns, focus on Contraction Ducts: 40 microns; Microtrabeculae diameter in chamber: 100 microns; Cavity periphery fence gap: 20 microns, the width on single hurdle: 80 microns; Whole cavity yardstick: about 15mm × 29mm; All structure heights are about 100 microns of these structures.In Ngatively pressurized sampling process, play a supporting role, prevent the obstruction of subsiding of emulsion droplet collecting zone.
The preparation of embodiment 2:PDMS chip
After making silicon chip, make individual layer and double-deck PDMS chip by moulding method.First the ratio that PDMS performed polymer is added to 10ml paraffin oil in every 200g fully mixes PDMS and paraffin oil, again the liquid phase after mixing and solidifying agent (mass ratio 10:1) are mixed, vacuumize degasification, be cast on above-mentioned mould, on slide glass, scumbling one deck is sneaked out the PDMS of paraffin oil in addition, leave standstill 1h, be put on 65 DEG C of hot plates Procuring 30 minutes, the PDMS being cast on mould is peeled off, punching, to there is the glass coating face after pipe surface and coating to fit together, and above sample introduction groove, add a cover a cover glass, in case Ngatively pressurized sampling groove sink. finally the PDMS posting is put on the hot plate of 85 DEG C and heat 10min, make its bonding.
Embodiment 3: the generation (Fig. 2) of emulsion droplet
PCR premixed liquid: comprise 10ul Roche 480 Probe Premix in 20ul premixed liquid, each 250nM EGFR gene 19 exon upstream and downstream primers, 200nM TaqMan probe, 10ng genomic dna.
First prepare PCR premixed liquid, inject PCR premixed liquid at injection port, oil injection port injects the whiteruss (containing 3%Abil EM90) containing emulsifying agent, syringe is placed on pump, dispensing end connects infusion strap, leather strap is inserted to suction orifice, tail end meets Simple negative-pressure pump (COSMO, Double Type 12000), pumping velocity moderate speed is set, sample size 15ul, utilize the suction function of negative pressure pump, PCR premixed liquid and paraffin oil are flowed towards outlet direction by injection port, at chip criss-cross construction place (Figure 1B), under the folder stream effect of both sides paraffin oil, cutting forms and receives the emulsion droplet (diameter 50 about μ m) of upgrading.Under the effect on micro-dam, (see Fig. 1 C, between Wei Ba dam, size is less than 20 μ m), and emulsion droplet is assembled concentrated in emulsion droplet collecting chamber, fills up cavity, now with PDMS sealing injection port and outlet.
Embodiment 4:PCR amplification
This chip can be put into PCR instrument and carry out the situ PCR (in situ PCR) at sheet, PCR cycling program is: 95 DEG C of 10min denaturations, 95 DEG C of 10s, 58 DEG C of 40s circulations, totally 40 circulations, last 4 DEG C of insulations, adopt fluorescence microscope result, CCD takes a picture, the counting positive emulsion droplet of fluorescent signal (Fig. 3).
Claims (8)
1. the making method of the digital pcr chip based on the saturated PDMS material of mineral oil, is characterized in that comprising the preparation of chip, generation and the pcr amplification of emulsion droplet,
(1) preparation of chip comprises design and the preparation of mould, first make microchannel and sample introduction groove, then be the preparation of PDMS material, the preparation of PDMS material is first to use uniform a certain amount of mineral oil PDMS monomer and solidifying agent mix and are cast on mould, in PDMS chip crosslinking curing process, mineral oil is filled space wherein automatically, the PDMS chip of making is to greatest extent engulfed oil phase in digital pcr system, maintains the stability of emulsion droplet and PCR reaction in PCR reaction process; In every 1ml PDMS, containing 100-400 μ l mineral oil, described mineral oil is whiteruss;
(2) generation of emulsion droplet
The formation of micro-fluidic chip emulsion droplet is by " ten " font or two kinds of chip structure forms of " T " font, and by adjusting oil phase and the size of water sample channel, the difference of length, size or concentration to formed emulsion droplet regulate and control; Emulsion droplet is made, is collected on same chip, then in the enterprising performing PCR amplification of same chip;
(3) pcr amplification
Chip complete sample introduction is lain on the PCR instrument with original position PCR function and is increased, the denaturation temperature of amplification and renaturation, amplification temperature all with emulsion droplet in the condition of the PCR mixed solution that wraps up identical, but proper extension soaking time.
2. by method claimed in claim 1, it is characterized in that:
1. described chip region comprises generating structure and emulsion droplet collection structure two portions of emulsion droplet;
2. described chip material is slide glass or cover glass.
3. by method claimed in claim 1, it is characterized in that it is first to utilize CAD software design pattern to print mask that microchannel and sample introduction groove are made, then utilize photoresist material SU8 2050 on silicon chip, to make microchannel and sample introduction groove structure, in Ngatively pressurized sampling process, play a supporting role, prevent the obstruction of subsiding of emulsion droplet collecting zone.
4. by method claimed in claim 1, it is characterized in that the making of described PDMS chip is:
1. the ratio that first PDMS performed polymer is added to 10ml paraffin oil in every 200g fully mixes PDMS and paraffin oil, then the mass ratio 10:1 liquid phase after mixing and solidifying agent are mixed, and vacuumizes degasification, is cast on mould;
2. on slide glass, scumbling one deck is the PDMS that sneaks out paraffin oil, leaves standstill 1h, is put on 65 DEG C of hot plates Procuring 30 minutes;
3. the PDMS being cast on mould is peeled off, punching, will have the glass coating face after pipe surface and coating to fit together, and add a cover a cover glass above sample introduction groove, in case Ngatively pressurized sampling groove sink;
4. on the last hot plate of the PDMS posting being put into 85 DEG C, heat 10min, make its bonding.
5. by method claimed in claim 1, it is characterized in that forming emulsion droplet by cross mode is:
1. inject PCR premixed liquid at sample feeding mouth, oily injection port injects the whiteruss containing 3%Abil EM90 containing emulsifying agent;
2. syringe is placed on pump, dispensing end connects infusion strap, and leather strap is inserted to suction orifice, and tail end connects negative pressure pump;
3. utilize the suction function of negative pressure pump, PCR premixed liquid and paraffin oil by injection port towards outlet direction flow, at chip criss-cross construction place, under the folder stream effect of both sides paraffin oil cutting form receive upgrading emulsion droplet;
4. under the effect on micro-dam, emulsion droplet is assembled concentrated in emulsion droplet collecting chamber, fills up cavity, now with PDMS sealing injection port and outlet;
Described PCR premixed liquid: comprise 10 μ l Roche480Probe Premix in 20 μ l premixed liquids, each 250nM EGFR gene 19 exon upstream and downstream primers, 200nM TaqMan probe, 10ng genomic dna.
6. by method claimed in claim 5, it is characterized in that:
A) 3. to cut the upgrading emulsion droplet diameter of receiving of formation be 50 μ m to step;
B) step 4. between described Wei Ba dam size be less than 20 μ m.
7. by method claimed in claim 1, while it is characterized in that the amplification of PCR original position, cycling program is 95 DEG C of 10min denaturations, 95 DEG C of 10s, and 58 DEG C of 40s circulate 40 times, last 4 DEG C of insulations.
8. by method claimed in claim 1, while it is characterized in that emulsion droplet generates:
1. inflow pipeline and sample channel height 20-120 μ m, width 20-150 μ m all can form good emulsion droplet;
2. the emulsion droplet diameter that cutting forms is skin upgrading or the upgrading of receiving, and it is stable that emulsion droplet diameter all can keep below 150 μ m.
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| Micropumps, microvalves, and micromixers within PCR microfluidic chips: Advances and trends;Chunsun Zhang et al;《Biotechnology Advances》;20071231(第25期);483-514 * |
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