CN105733922B - Integrated micro-valve micro-fluidic chip and preparation method thereof for ultrafast nucleic acid amplification - Google Patents
Integrated micro-valve micro-fluidic chip and preparation method thereof for ultrafast nucleic acid amplification Download PDFInfo
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Abstract
The integrated micro-valve micro-fluidic chip and preparation method thereof that the present invention provides a kind of for ultrafast nucleic acid amplification, the chip use Multi-layer design, mainly include PCR conversion zone, PDMS film layer, heating fluid channel layer and gas passage layers；After oxygen plasma treatment, key is sealed on support glass each layer together with；Temperature needed for the chip controls nucleic acid amplification using integrated micro-valve is adjusted, and by the fluid by temperature has been set according to set program, under the control of integrated micro-valve, the heating zone micro-fluidic chip PCR is passed sequentially through, to realize overall process needed for nucleic acid amplification.The chip is dimethyl silicone polymer (PDMS) using material, and preparation method is prepared using the method for soft lithographic.The method of the present invention temperature stability it is high, it can be achieved that nucleic acid amplification temperature ultrafast switching, detected in medicine, the fields such as monitoring of infectious disease are with a wide range of applications.
Description
Technical field
The invention belongs to the application fields such as micro fluidic chip technical field and detection of nucleic acids, and in particular to a kind of ultrafast core
The integrated micro-valve micro-fluidic chip and preparation method thereof of acid amplification.
Background technique
Polymerase chain reaction (PCR) is the isothermal DNA amplification that the mid-80 grows up, it has special, quick
Outstanding advantages of sense, yield high, quick, easy, reproducible, easy automation, it is widely used in Bacteria Detection, genetic disease is examined
The fields such as disconnected, diagnosing tumor, forensic identification.Round pcr is a revolutionary invention and milestone in field of biomedicine.It passes
The PCR reaction system of system is larger, and reagent consumption is big, detection it is at high cost, simultaneously because system system used is big, therefore PCR
The thermal capacitance of system is big, therefore warming and cooling rate is restricted, and the time of entire PCR reaction is mostly more than 1 hour.
In recent years, in flow control research field, temperature needed for having researcher and realizing PCR amplification using different temperature control strategies
Degree conversion, substantially increases the reaction speed of PCR.It is reacted with continuous flow type micro-fluidic chip PCR for representative, uses driving
PCR reaction mixture completes the entire mistake of the PCR reaction such as denaturation, annealing and extension by two or three different temperatures regions
Journey.In continuous flow pcr chip, when reaction solution flows through different warm areas, automatic temperature changing, rate is fast, large specific surface area, the reaction time
It is short.24 amplification cycles may be implemented in 5 minutes.However, realizing that PCR expands in flow process due to continuous flow type PCR system
Increase, brings difficulty to the detection in amplification procedure, can not achieve widely used real-time quantitative PCR reaction system.Therefore, real
Existing one kind can accelerate PCR reaction speed and the new microfluidic chip system tool in conjunction with existing real-time quantitative PCR system
It is significant.
Summary of the invention
The object of the present invention is to provide a kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, which is solved
The problems such as PCR process thermal capacitance is big, and warming and cooling rate is slow.
The integrated micro-valve micro-fluidic chip and preparation method thereof that the present invention provides a kind of for ultrafast nucleic acid amplification, specifically
Technical solution is as follows:
A kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, the chip use Multi-layer design, mainly include
PCR conversion zone, PDMS film layer, heating fluid channel layer and gas passage layers；Each layer after oxygen plasma treatment, key and
Together, it and is sealed on support glass；
It uniformly arranges in the PCR conversion zone 9~100 PCR reaction solution ponds；
The PDMS film layer is surface without structure, with a thickness of 10~100 microns of PDMS film；
The 3 heating fluid channel layer includes 6 heating fluid inlets, 7 heating channels, 8 heated fluid outlets, heating channel
For the fold-line-shaped of detour, heats there are two one end of the channel or three heating fluid inlets, the other end have a heated fluid outlet；
The gas passage layers include two or 3 10 gas passages, and 10 gas passage one end are the 9 gas access other ends
For gas micro-valve；
PCR conversion zone is top layer, is secondly PDMS film layer, heating fluid channel layer and gas passage layers, heating stream
The heating channel of body channel layer is located immediately at the underface of PCR conversion zone PCR reaction tank, the gas micro-valve and heat of gas passage layers
The heating channel of fluid channel layer is corresponding.
The switching of each temperature needed for the chip realizes nucleic acid amplification by integrated micro-valve.
The opening and closing of the micro-valve is closed and the duration is by process control.
The heating fluid that the chip is controlled is water, air or mineral oil etc..
The PCR conversion zone can be reaction tank, or reaction channel, form can according to need design.
A kind of preparation method of the integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, this method include following step
It is rapid:
First with photoresist SU8 prepares each layer template: gas passage layers template, heating fluid channel layer and heating fluid layer
Template and PCR conversion zone template；
Then after aggressiveness before the PDMS of volume ratio 10:1 and crosslinking agent being mixed degassing, rotary coating is in gas passage template
With heating channel template, it is placed in polymerization in 80 DEG C of baking ovens, gas passage layers, heating fluid channel layer is respectively prepared；
After aggressiveness before the PDMS of volume ratio 10:1 and crosslinking agent are mixed degassing, PCR reaction is prepared by the way of casting
Layer；Make its polymerization in 80 DEG C of baking ovens；PDMS film layer directlys adopt the PDMS film of commercialization；
The PDMS having polymerize is top layer according to PCR conversion zone, is secondly PDMS film layer, and heating fluid channel layer is gentle
The sequence of body channel layer, after oxygen plasma treatment, key is sealed on support glass together with, completes integrated micro-valve
The preparation of micro-fluidic chip.
Provided by the present invention for the integrated micro-valve micro-fluidic chip of ultrafast nucleic acid amplification, the integrated micro-valve is micro-fluidic
Chip material therefor is dimethyl silicone polymer (PDMS), and preparation method is prepared using the method for soft lithographic.
Provided by the present invention for the integrated micro-valve micro-fluidic chip of ultrafast nucleic acid amplification, the fluid of steady temperature can be with
For water, air, mineral oil etc..
It is each required for nucleic acid amplification provided by the present invention for the integrated micro-valve micro-fluidic chip of ultrafast nucleic acid amplification
Temperature is controlled by integrated micro-valve, and the switching time between temperature is determined by the opening/closing time of micro-valve, the duration of each temperature
It is adjustable.The opening and closing of micro-valve is closed and the duration is by process control.
Provided by the present invention for the integrated micro-valve micro-fluidic chip of ultrafast nucleic acid amplification, it is easy to and other micro-fluidic chips
Integration ofTechnology realizes the fully integrated of nucleic acid amplification.Such as: it can be directly integrated nucleic acid extraction unit, realize nucleic acid purification process
With the micro-fluidic chip of amplification one；With the integrated quick detection that drop digital pcr may be implemented of drop digital pcr.
Provided by the present invention for the integrated micro-valve micro-fluidic chip of ultrafast nucleic acid amplification, a variety of conditions and more may be implemented
It detects, while can also directly be measured with real-time quantitative while a sample.
Provided by the present invention for the integrated micro-valve micro-fluidic chip of ultrafast nucleic acid amplification, the advantage is that:
1, the control of PCR temperature is carried out using the fluid of fixed temperature, temperature fluctuation is small；
2, temperature control does not need complicated temperature controlling instruments；
3, temperature switches fast (< 1s), it can be achieved that ultrafast nucleic acid amplification；
4, integrated microelectrode is not needed, it is at low cost.
5, the amplification method is easy to integrated with microflow control techniques such as extractions, realizes nucleic acid amplification automation.
Detailed description of the invention
The structural representation of the nucleic acid amplification chip of Fig. 1 integrated micro-valve；(a) schematic diagram of a layer structure is reacted for PCR, (b) is
PDMS film structural schematic diagram (c) is heating fluid channel schematic diagram of a layer structure；It (d) is gas circuit schematic diagram of a layer structure；(e) it is
The schematic diagram of entire chip；
Wherein: 1PCR conversion zone, 2PDMS film, 3 heating fluid channel layers, 4 gas circuit layers, 5 be PCR reaction solution pond, and 6 are
Heating fluid inlet, 7 be heating channel, and 8 be heated fluid outlet, and 9 be gas access, and 10 be gas passage, and 11 is micro- for gas
Valve,.
Fig. 2 integrated micro-valve nucleic acid amplification chip material object photo；
The combination chip photo of Fig. 3 integrated micro-valve nucleic acid amplification and drop digital pcr；
Fig. 4 integrated micro-valve temperature switching characterization；
The chip electrophoresis of Fig. 5 integrated micro-valve Rapid nucleic acid amplification characterizes；
The amplification of Fig. 6 integrated micro-valve Rapid nucleic acid is used for identification；
Specific embodiment
Following embodiment will be further described the present invention, but not thereby limiting the invention.
Aggressiveness and crosslinking agent use the sylgard 184 of Dow Corning Corporation before PDMS in the embodiment of the present invention.
A kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, the chip use Multi-layer design, mainly include
PCR conversion zone 1, PDMS film layer 2, heating fluid channel layer 3 and gas passage layers 4；Each layer is after oxygen plasma treatment, key
Together, it and is sealed on support glass；
It uniformly arranges in the PCR conversion zone 1 9~100 PCR reaction solution ponds 5；
The PDMS film layer 2 is surface without structure, with a thickness of 10~100 microns of PDMS film；
The heating fluid channel layer 3 includes heating fluid inlet 6, heats channel 7, and heated fluid outlet 8 heats channel
7 be the fold-line-shaped of detour, and there are two heating 7 one end of channel or three heating fluid inlets 6, the other end have a heating fluid to go out
Mouth 7；
The gas passage layers 4 include two or 3 gas passages 10, and 10 one end of gas passage is that 9 gas accesses 9 are another
End is gas micro-valve 11；
PCR conversion zone 1 is top layer, is secondly PDMS film layer 2, heating fluid channel layer 3 and gas passage layers 4, adds
The heating channel 7 of zone of heat liberation layer 3 is located immediately at the underface of PCR conversion zone 1PCR reaction tank 5, the gas of gas passage layers 4
Body micro-valve 11 is corresponding with the heating channel 7 of zone of heat liberation layer 3.
The switching of each temperature needed for the chip realizes nucleic acid amplification by integrated micro-valve.
The opening and closing of the micro-valve is closed and the duration is by process control.
The heating fluid that the chip is controlled is water, air or mineral oil etc..
The PCR conversion zone can be reaction tank, or reaction channel, form can according to need design.
Embodiment 1
The structural unit of the nucleic acid amplification chip of integrated micro-valve
A kind of preparation method of the integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, this method include following step
It is rapid:
First with photoresist SU8 prepares each layer template: gas passage layers template, heating fluid channel layer and heating fluid layer
Template and PCR conversion zone template；
Then after aggressiveness before the PDMS of volume ratio 10:1 and crosslinking agent being mixed degassing, rotary coating is in gas passage template
With heating channel template, it is placed in polymerization in 80 DEG C of baking ovens, gas passage layers, heating fluid channel layer is respectively prepared；
After aggressiveness before the PDMS of volume ratio 10:1 and crosslinking agent are mixed degassing, PCR reaction is prepared by the way of casting
Layer；Make its polymerization in 80 DEG C of baking ovens；PDMS film layer directlys adopt the PDMS film of commercialization；
The PDMS having polymerize is top layer according to PCR conversion zone, is secondly PDMS film layer, and heating fluid channel layer is gentle
The sequence of body channel layer, after oxygen plasma treatment, key is sealed on support glass together with, completes integrated micro-valve
The preparation of micro-fluidic chip.
As shown in Figure 1, the integrated chip part is broadly divided into PCR reaction zone, PDMS film, heating fluid channel layer is gentle
Body channel layer.Entire channel is the preparation of PDMS material, wherein the part of gas passage micro-valve, in the case where pressure, upward shape
Become, form the pressure to fluid path channel, the on-off of the heating fluid of micro-valve may be implemented.Fluid heats channel and PCR reaction tank
Between between alternating floor PDMS film, in favor of the quick transmitting of heat.The steady temperature fluid that chip is uses water, air or mine
Object oil etc..Fig. 2 is the photo of simple integrated micro-valve micro-fluidic chip.The chip can also be with micro-fluidic other technologies knot simultaneously
It closes, forms more complicated micro-fluidic PCR detection chip.Fig. 3 is that micro-valve integrated chip is micro-fluidic in conjunction with digital drop round pcr
Chip, the integrated chip have a PCR drop fixed cell more than 10,000, the quick detection of drop digital pcr may be implemented.
Embodiment 2
Integrated micro-valve temperature switching characterization
In such a way that micro-valve control is different with different dyes fluid, acidity can be switched to the chip temperature and carry out table
Sign.The video recording of switching fluid is recorded using CCD digital camera, as shown in Figure 4.The chip can realize entire chip in 0.5s
The switching of different dyes fluid, therefore fast PCR reaction may be implemented.
Embodiment 3
The chip electrophoresis of integrated micro-valve Rapid nucleic acid amplification characterizes and application
Rapid nucleic acid amplification is carried out by the chip, chip electrophoresis separation is carried out to its product, amplified production is tested
Card.The nucleic acid amplification of 35 circulations may be implemented in the chip in 10 minutes.Chip electrophoresis figure is as shown in Figure 5.The chip simultaneously
Being combined with electrophoretic separation can also be applied to legal medical expert's identification, and the STR testing result of practical blood sample is as shown in Figure 6.
Claims (4)
1. a kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, which is characterized in that the chip uses Multi-layer design,
It mainly include PCR conversion zone, PDMS film layer, heating fluid channel layer and gas passage layers；Each layer is through oxygen plasma treatment
Afterwards, key is together with, and is sealed on support glass；
It uniformly arranges in the PCR conversion zone 9~100 PCR reaction solution ponds；
The PDMS film layer is surface without structure, with a thickness of 10~100 microns of PDMS film；
The heating fluid channel layer includes heating fluid inlet, heats channel, heated fluid outlet, and heating channel is detour
Fold-line-shaped, there are two heating one end of the channel or three heating fluid inlets, the other end have a heated fluid outlet；
The gas passage layers include two or 3 gas passages, and gas passage one end is that the gas access other end is that gas is micro-
Valve；
PCR conversion zone is top layer, is secondly PDMS film layer, heating fluid channel layer and gas passage layers, and heating fluid is logical
The heating channel of channel layer is located immediately at the underface of PCR conversion zone PCR reaction tank, the gas micro-valve and hot fluid of gas passage layers
The heating channel of channel layer is corresponding；
The switching of each temperature needed for the chip realizes nucleic acid amplification by integrated micro-valve.
2. the integrated micro-valve micro-fluidic chip described in accordance with the claim 1 for ultrafast nucleic acid amplification, it is characterised in that micro-valve
Opening and closing close and the duration by process control.
3. the integrated micro-valve micro-fluidic chip described in accordance with the claim 1 for ultrafast nucleic acid amplification, which is characterized in that the core
The heating fluid that piece is controlled is water, air or mineral oil.
4. the preparation method of the integrated micro-valve micro-fluidic chip described in accordance with the claim 1 for ultrafast nucleic acid amplification, special
Sign is that this method comprises the following steps:
First with photoresist SU8 prepares each layer template: gas passage layers template, heating fluid channel layer and heating fluid layer template
With PCR conversion zone template；
Then after aggressiveness before the PDMS of volume ratio 10:1 and crosslinking agent being mixed degassing, rotary coating is in gas passage template and adds
Passage of heat template is placed in polymerization in 80 DEG C of baking ovens, gas passage layers, heating fluid channel layer is respectively prepared；
After aggressiveness before the PDMS of volume ratio 10:1 and crosslinking agent are mixed degassing, PCR conversion zone is prepared by the way of casting；?
Make its polymerization in 80 DEG C of baking ovens；PDMS film layer directlys adopt the PDMS film of commercialization；
The PDMS having polymerize is top layer according to PCR conversion zone, is secondly PDMS film layer, and heating fluid channel layer and gas are logical
The sequence of channel layer, after oxygen plasma treatment, key is sealed on support glass together with, completes integrated micro-valve miniflow
Control the preparation of chip.
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| CN107619781B (en) * | 2016-07-15 | 2022-01-25 | 中国检验检疫科学研究院 | Single-reaction temperature-control high-throughput micro-fluidic chip nucleic acid amplification device |
| CN106867882B (en) * | 2017-02-09 | 2021-04-06 | 南京大学 | Multi-chamber microfluidic PCR chip for aptamer screening and manufacturing method and application thereof |
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