CN103343092A - 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 the making method of a kind of digital pcr based on the saturated PDMS material of mineral oil (polymerase chain reaction) chip, the chip of making.Be used for low abundance detection of nucleic acids, can be applied to fields such as biology, medical science and environmental science.
Background technology
(Fluorescence Quantitative Polymerase Chain Reaction, FQ-PCR qPCR) have developed into the routine techniques of a key of biology field to quantitative fluorescent PCR, have greatly promoted the development of life science every field.But, the factor that influences its amplification efficiency in the pcr amplification process is a lot, be difficult to guarantee that actual sample is identical with amplification efficiency between standard model and the different sample, (Cycling Threshold Value is not invariable Ct) to cause basis-cycle threshold that its quantitative analysis relies on thus.Therefore qPCR's is " relative quantification " quantitatively, and its accuracy and circulation ratio still can not satisfy the requirement of molecular biology quantitative analysis.In addition, owing 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 the somatocyte at present.
(Digital PCR is a kind of nucleic acid quantification method of counting based on the single-molecule PCR method dPCR) to digital pcr, 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 behind the 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 the 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 not have fluorescent signal.According to the volume of relative proportion and reactor, just can extrapolate the nucleic acid concentration of original solution.Different with traditional quantitative PCR, digital pcr can be realized the absolute quantitation of initiate dna template by the method for direct census.
In addition, digital pcr still is a kind of method that can identify micro-mutant in a large amount of wild-type DNA backgrounds.Because the digital pcr technology can be separated the template DNA molecule separately in advance and increase, this has just been avoided the amplification inhibition of high abundance allelotrope nucleic acid to variant nucleic acid, has therefore improved the detector efficiency of micro-variant nucleic acid.Emulsion droplet digital pcr technology can detect and be low to moderate 0.001% sudden change fragment, 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 at first by manual stepwise dilution and distribute sample to microwell plate, then microwell plate is placed the enterprising performing PCR reaction of thermal cycler, reaction finishes the back and reads fluorescent signal in each micropore by instrument, finally according to the ratio of Poisson's distribution principle and positive droplet, binding analysis software can calculate concentration or the copy number that provides target molecule to be checked.This mode complex operation, flux is little, efficient is low, and less drop decomposes the dynamicrange that number has also limited its precision and can survey, and application facet has great limitation.In recent years, new vitality has been injected in the development that develops into the digital pcr technology of microflow control technique.Because microflow control technique in the advantage of microfluid aspect controlling, makes us decomposed sample can be risen even skin is upgraded for receiving, and obtains more decomposed sample number, thereby improves detection sensitivity, confidence level and the dynamicrange degree of digital pcr technology greatly.In addition, microflow control technique automatization, easy of integration, advantage that flux is high also can greatly improve the detection efficiency of digital pcr technology.
In the advantage of microfluid aspect controlling, existing a plurality of research groups and company have developed the digital pcr system based on microflow control technique on the recent international by microflow control technique.More typically comprise the BioMark based on the microchamber type that Fluidigm company releases
TMThe QX100TM system based on the 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 automation of operation, detection and can survey the concentration dynamicrange and all be greatly improved, but still there is certain defective in actual applications, 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 increased the cost of system, limited its application.
PDMS(polydimethylsiloxane, polydimethylsiloxane) is a kind of rubber-like high molecular polymer, because of its cost low, use simple, and has a good optical characteristic, good insulation performance, characteristics such as good unreactiveness and air permeability and good become a kind of polymer materials that is widely used in field such as micro-fluidic.
In the digital pcr field, more people adopt PDMS to make emulsion droplet and generate chip, directly less at the example of PDMS chip enterprising line number word pcr amplification and analysis, major cause is because one comes ventilation property strong, two because it has good lipophilicity, therefore the receptivity superpower to having of oiliness molecule in the PCR thermal cycling process can't realize amplification.
Also had minority directly to carry out the example of digital pcr amplification in recent years on the PDMS chip, they avoid the mode of oil, liquid volatilization to be mainly: put on the 1:PDMS chip emulsion droplet dot matrix and stick sheet glass; 2: spraying is to polyxylene in the chip.
First kind of mode recited above can't stop PDMS chip material itself to the mineral pick up the oil at all, and PDMS itself is very strong to the receptivity of mineral oil; Described second way processing condition more complicated, common laboratory can't satisfy this condition.
In sum, the present invention intends disclosing a kind of digital pcr chip preparation method based on the saturated PDMS material of mineral oil.The present invention has overcome because the emulsion droplet wild effect that PDMS oil receptivity causes by force, and compares with present existing digital pcr chip technology, and it is low to have a cost, and advantages of simple operation, application prospect are very extensive.
Summary of the invention
The objective 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 the 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.The material of described chip preparation 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
Because the thermal cycling process of a strictness of PCR reaction needed, so the characteristics of the good permeability of PDMS own become 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 the PCR reaction solution be dispersed into a plurality of thousands of PCR reaction members independently automatically in the middle of oil phase, independently carries out separately on the basis that PCR reacts.Therefore, the sorption of the oil phase of PDMS will form tremendous influence to the stability of each PCR reaction member.The preparation of chip of the present invention comprises Mould design and preparation, make microchannel and sample introduction groove earlier, be the preparation of PDMS material then, the PDMS material preparation is (containing the described mineral oil of 100-400 μ l mineral oil among every 1ml PDMS is whiteruss) in the PDMS monomer of using earlier a certain amount of mineral oil, in PDMS chip crosslinking curing process, mineral oil is filled space wherein automatically, therefore can suppress the PDMS chip made to greatest extent to the engulfing of oil phase in the digital pcr system, keep the stability of emulsion droplet and PCR reaction in the PCR reaction process.
2, the generation of emulsion droplet
The formation of micro-fluidic chip emulsion droplet can be by " 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 situations such as the size of formation emulsion droplet, concentration.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, emulsion droplet is made, collected at same chip; Then in the enterprising performing PCR amplification of same chip.
3, pcr amplification
The chip that sample introduction is finished lies on the PCR instrument with original position PCR function and increases, 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 the soaking time proper extension.
Making method of the present invention and conventional digital pcr method ratio, consumables cost is low, emulsion droplet formation condition and observational technique is easy as a result, required testing installation common laboratory can satisfy.
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) is prepared PDMS digital pcr chip, chip comprises emulsion droplet generating structure, emulsion droplet collection structure two portions.Emulsion droplet is made, is collected at same chip, then in the enterprising performing PCR amplification of same chip.This chip has been avoided engulfing of oil phase in the digital pcr system of PDMS, is conducive to keep the stability of emulsion droplet and PCR reaction in the PCR reaction process.The present invention has following advance:
[1] the present invention has overcome because the emulsion droplet wild effect that PDMS oil receptivity causes by force;
The PDMS chip material of ordinary numbers pcr chip can be engulfed emulsion droplet and form necessary oil phase in the thermal cycling process, cause the emulsion droplet instability in the chip, even causes the aqueous phase liquid volatilization and the amplification failure.The present invention fills hole among the PDMS with paraffin oil, thereby has suppressed its phagolysis to oil phase, has safeguarded the stability of emulsion droplet in the pcr amplification process, for the success of pcr amplification provides guarantee.
[2] oily saturated PDMS changes the PDMS surface properties, can reduce chip to the adsorptive power of enzyme, nucleic acid, fluorescence dye etc., the trouble of having avoided the chip surface treatment step to bring;
Because the high water transport property of PDMS material surface reaches 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 the absorption biological respinse as the biomacromolecule of albumen, nucleic acid etc. and fluorescence dye, and therefore application is very extensive in biological respinse.The present invention can reach the effect of PDMS being carried out surface modification simultaneously with the hole among the paraffin oil filling PDMS, and the saturated PDMS material of paraffin oil has further guaranteed the stability of testing to not absorption substantially such as enzyme, nucleic acid, fluorescence dye.
[3] digital pcr method involved in the present invention is compared with present digital pcr chip technology, and cost is low, and is easy and simple to handle, and routine test equipment can satisfy the test needs.
At present commercially available digital pcr chip to be adopted special plant and instrument generation emulsion droplet and reads 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 the emulsion droplet making, and data read adopts 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 at same chip, so this method cost is low, and be simple and easy to do, is easy to promote.
Description of 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 synoptic diagram;
A: emulsion droplet generating principle synoptic diagram; The B:PCR amplification is analyzed synoptic diagram;
Fig. 3: oily saturated PDMS digital pcr chip detection result;
The 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 characteristics of the present invention and marked improvement with specific embodiment below, but the present invention only is confined to embodiment by no means.
Embodiment 1: Mould design and making (Fig. 1)
The present invention has studied various chips structure and positive Ngatively pressurized sampling emulsion droplet has been formed the influence of stability, find at last 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 employing cross clamp stream negative pressure mode generation emulsion droplet.At first utilize CAD software design pattern to print mask, utilize photoresist material SU8 2050 to make microchannel and sample introduction groove structure (see figure 1) at silicon chip then, oil phase sample channel width: 200 microns, PCR mixed solution sample channel: 60 microns, focus on and shrink pipeline: 40 microns; Microtrabeculae diameter in the 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 these structures.In the 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
Make individual layer and double-deck PDMS chip by moulding method after making silicon chip.At first the PDMS performed polymer is added the ratio of 10ml paraffin oil in every 200g with PDMS and the abundant mixing of paraffin oil, again the liquid phase behind the mixing and solidifying agent (mass ratio 10:1) are mixed, vacuumize degasification, be cast on the above-mentioned mould, scumbling one deck is sneaked out the PDMS of paraffin oil on slide glass in addition, leave standstill 1h, be put on 65 ℃ of hot plates Procuring 30 minutes, the PDMS that is cast on the mould is peeled off, punching will have the glass coating face after pipe surface and the coating to fit together, and add a cover a cover glass above the sample introduction groove, in case the Ngatively pressurized sampling groove sink. at last the PDMS that posts is put on 85 ℃ the hot plate and heat 10min, make its bonding.
Embodiment 3: the generation of emulsion droplet (Fig. 2)
PCR premixed liquid: comprise 10ul Roche 480 Probe Premix in the 20ul premixed liquid, each 250nM EGFR gene 19 exon upstream and downstream primer, 200nM TaqMan probe, 10ng genomic dna.
At first prepare the PCR premixed liquid, inject the PCR premixed liquid at injection port, the oil injection port injects the whiteruss (containing 3%Abil EM90) that contains emulsifying agent, syringe is placed on the pump, dispensing end connects the transfusion leather strap, leather strap is inserted suction orifice, tail end meets simple and easy negative pressure pump (COSMO, Double Type 12000), the 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 the outlet direction by injection port, at chip criss-cross construction place (Figure 1B), form in cutting under the folder stream effect of both sides paraffin oil and to receive the emulsion droplet (about the about 50 μ m of diameter) of upgrading.Under the effect on little dam (see Fig. 1 C, size is less than 20 μ m between the Wei Ba dam), emulsion droplet is assembled concentrated in the emulsion droplet collecting chamber, fills up cavity, and seal injection port and outlet with PDMS this moment.
Embodiment 4:PCR amplification
This chip can be put into the PCR instrument carry out original position pcr amplification (in situ PCR) at sheet, the PCR cycling program is: 95 ℃ of pre-sex change of 10min, 95 ℃ of 10s, 58 ℃ of 40s circulations, totally 40 circulations, the fluorescence microscope result is adopted in last 4 ℃ of insulations, CCD takes a picture, the positive emulsion droplet (Fig. 3) of counting fluorescent signal.
Claims (8)
1. based on the making method of the digital pcr chip of the saturated PDMS material of mineral oil, it is characterized in that comprising the preparation of chip, generation and the pcr amplification of emulsion droplet,
(1) preparation of chip comprises Mould design and preparation, make microchannel and sample introduction groove earlier, be the preparation of PDMS material then, the PDMS material preparation is in the PDMS monomer of using earlier a certain amount of mineral oil, in PDMS chip crosslinking curing process, mineral oil is filled automatically space wherein, and the PDMS chip of making is to greatest extent engulfed oil phase in the digital pcr system, keeps the stability that emulsion droplet and PCR react in the PCR reaction process; Contain 100-400 μ l mineral oil among every 1ml PDMS, described mineral oil is whiteruss;
(2) generation of emulsion droplet
The formation of micro-fluidic chip emulsion droplet, is regulated and control size or the concentration of formation emulsion droplet by adjusting oil phase and the size of water sample channel, the difference of length by " ten " font or two kinds of chip structure forms of " T " font; Emulsion droplet is made, is collected at same chip, then in the enterprising performing PCR amplification of same chip;
(3) pcr amplification
The chip that sample introduction is finished lies on the PCR instrument with original position PCR function and increases, 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 the described method of 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 the described method of claim 1, it is characterized in that it is at first to utilize CAD software design pattern to print mask that microchannel and sample introduction groove are made, utilize photoresist material SU8 2050 to make microchannel and sample introduction groove structure at silicon chip then, in the Ngatively pressurized sampling process, play a supporting role, prevent the obstruction of subsiding of emulsion droplet collecting zone.
4. by the described method of claim 1, it is characterized in that the making of described PDMS chip is:
1. at first the PDMS performed polymer is added the ratio of 10ml paraffin oil in every 200g with PDMS and the abundant mixing of paraffin oil, again the mass ratio 10:1 liquid phase behind the mixing and solidifying agent are mixed, vacuumize degasification, be cast on the mould;
2. scumbling one deck is the PDMS that sneaks out paraffin oil on slide glass, leaves standstill 1h, is put on 65 ℃ of hot plates Procuring 30 minutes;
3. the PDMS that will be cast on the mould peels off, and punching will have the glass coating face after pipe surface and the coating to fit together, and add a cover a cover glass above the sample introduction groove, in case the Ngatively pressurized sampling groove is sagging;
4. at last the PDMS that posts is put on 85 ℃ the hot plate and heat 10min, make its bonding.
5. by the described method of claim 1, it is characterized in that forming emulsion droplet by cross mode is:
1. inject the PCR premixed liquid at the sample feeding mouth, oily injection port injects the whiteruss that contains 3%AbilEM90 that contains emulsifying agent;
2. syringe is placed on the pump, dispensing end connects the transfusion leather strap, and leather strap is inserted suction orifice, and tail end connects negative pressure pump;
3. utilize the suction function of negative pressure pump, PCR premixed liquid and paraffin oil are flowed towards the outlet direction by injection port, and at chip criss-cross construction place, cutting forms and receives the emulsion droplet of upgrading under the folder stream effect of both sides paraffin oil;
4. under the effect on little dam, emulsion droplet is assembled concentrated in the emulsion droplet collecting chamber, fills up cavity, and seal injection port and outlet with PDMS this moment;
Described PCR premixed liquid: comprise 10ul Roche 480 Probe Premix in the 20ul premixed liquid, each 250nM EGFR gene 19 exon upstream and downstream primer, 200nM TaqMan probe, 10ng genomic dna.
6. by the described method of 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 less than 20 μ m;
C) step 2. described negative pressure pump be COSMO, two models 12000.
7. by the described method of claim 1, cycling program is 95 ℃ of pre-sex change of 10min when it is characterized in that the amplification of PCR original position, 95 ℃ of 10s, and 58 ℃ of 40s circulate last 4 ℃ of insulations 40 times.
8. by the described method of claim 1, when 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 of cutting formation is skin upgrading or the upgrading of receiving, and it is stable that the emulsion droplet diameter all can keep below 150 μ m.
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