CN105733922A - Integrated microvalve micro-fluidic chip used for ultrafast nucleic acid amplification and preparation method of integrated microvalve micro-fluidic chip - Google Patents
Integrated microvalve micro-fluidic chip used for ultrafast nucleic acid amplification and preparation method of integrated microvalve micro-fluidic chip Download PDFInfo
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Abstract
The invention provides an integrated microvalve micro-fluidic chip used for ultrafast nucleic acid amplification and a preparation method of the integrated microvalve micro-fluidic chip. The integrated microvalve micro-fluidic chip adopts the multi-layer design, mainly comprising a PCR reaction layer, a PDMS film layer, a heating fluid passage layer and a gas passage layer; after oxygen plasma treatment, all the layers are bonded together, and connected to support glass in a sealed manner; the integrated microvalve micro-fluidic chip adopts an integrated microvalve to control the temperature regulation needed by nucleic acid amplification, fluid with the set temperature sequentially passes through PCR heating areas of the micro-fluidic chip according to the set procedure and under the control of the integrated microvalve, and thus the whole process needed by nucleic acid amplification is realized. The integrated microvalve micro-fluidic chip is made of polydimethylsiloxane (PDMS), and prepared through a soft-lithography method. According to the preparation method, the temperature stability is high, the ultrafast switching of the nucleic acid amplification temperature can be realized, and the integrated microvalve micro-fluidic chip has wide application prospects in the fields of medical testing, infectious disease surveillance and the like.
Description
Technical field
The invention belongs to the application such as micro fluidic chip technical field and detection of nucleic acids, be specifically related to integrated micro-valve micro-fluidic chip of a kind of ultrafast nucleic acid amplification and preparation method thereof.
Background technology
Polymerase chain reaction (PCR) is the isothermal DNA amplification that the mid-80 grows up, it has the outstanding advantages such as special, sensitive, productivity is high, quick, easy, reproducible, easy automatization, is widely used in the fields such as Bacteria Detection, diagnosis of genetic disorders, diagnosing tumor, forensic identification.Round pcr is a revolutionary invention in biomedical sector and milestone.Traditional PCR reaction system is relatively big, and reagent consumption is big, and the cost of detection is high, simultaneously because system system used is big, therefore the thermal capacitance of PCR system is big, and therefore warming and cooling rate is restricted, and the time of whole PCR reaction is mostly more than 1 hour.
In recent years, in stream control research field, there is researcher to adopt different temperature control strategies to realize the temperature transition needed for pcr amplification, substantially increase the response speed of PCR.Reacting for representative with continuous flow type micro-fluidic chip PCR, it adopts driving PCR reaction mixture by two or three different temperatures regions, completes the whole process of the PCR reactions such as degeneration, annealing and extension.In stream pcr chip continuously, when reactant liquor flows through different warm area, automatic temperature changing, speed is fast, and specific surface area is big, and the response time is short.24 amplification cycles can be realized in 5 minutes.But, due to continuous flow type PCR system, flow process realizes pcr amplification, the detection in amplification procedure is brought difficulty, it is impossible to realize widely used real-time quantitative PCR reaction system.Therefore, it is achieved one can accelerate PCR response speed, the new microfluidic chip system being combined with existing real-time quantitative PCR system again is significant.
Summary of the invention
It is an object of the invention to provide a kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, it is big that this chip solves PCR process thermal capacitance, the problems such as warming and cooling rate is slow.
The invention provides a kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification and preparation method thereof, concrete technical scheme is as follows:
A kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, this chip adopts Multi-layer design, mainly includes PCR conversion zone, PDMS film layer, heating fluid channel layer and gas passage layers；Each layer after oxygen plasma processes, key with together with, and be sealed in support glass；
In described PCR conversion zone, uniformly 9～100 PCR of arrangement react liquid pool；
Described PDMS film layer is surface non-structure, and thickness is the PDMS film of 10～100 microns；
Described 3 heating fluid channel layers include 6 heating fluid inlet, and 7 add the passage of heat, 8 heated fluid outlet, and adding the passage of heat is roundabout fold-line-shaped, and heating passage end has two or three heating fluid inlet, and the other end has a heated fluid outlet；
Described gas passage layers includes two or 3 10 gas passages, and 10 gas passage one end are the 9 gas access other ends is the micro-valve of gas；
PCR conversion zone is the superiors, secondly it is PDMS film layer, heating fluid channel layer and gas passage layers, the passage of heat that adds of heating fluid channel layer is located immediately at the underface of PCR conversion zone PCR reaction tank, the micro-valve of gas of gas passage layers and zone of heat liberation layer to add the passage of heat corresponding.
Described chip realizes the switching of nucleic acid amplification each temperature required by integrated micro-valve.
The opening and closing of described micro-valve is closed and the persistent period is by programme-control.
What described chip controlled adds hot fluid is water, air or mineral wet goods.
Described PCR conversion zone can be reaction tank, it is also possible to for reaction channel, its form can design as required.
The preparation method of a kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, the method comprises the steps:
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 aggressiveness before the PDMS of volume ratio 10:1 and cross-linking agent are mixed degassed after, rotary coating is in gas passage template and adds passage of heat template, is placed in 80 DEG C of baking ovens polymerization, is respectively prepared gas passage layers, heating fluid channel layer；
After degassed to aggressiveness before the PDMS of volume ratio 10:1 and cross-linking agent mixing, the mode of cast is adopted to prepare PCR conversion zone；It is made to be polymerized in 80 DEG C of baking ovens；PDMS film layer, directly adopts commercial PDMS film；
The PDMS being polymerized is the superiors according to PCR conversion zone, is secondly PDMS film layer, the order of heating fluid channel layer and gas passage layers, after oxygen plasma processes, key with together with, and be sealed in support glass, complete the preparation of integrated micro-valve micro-fluidic chip.
Integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification provided by the invention, described integrated micro-valve micro-fluidic chip material therefor is polydimethylsiloxane (PDMS), and preparation method adopts the method for soft lithographic to prepare.
Integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification provided by the invention, the fluid of its steady temperature can be water, air, mineral wet goods.
Integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification provided by the invention, each temperature required for nucleic acid amplification is by integrated micro-valve control, and the switching time between temperature is determined by the opening/closing time of micro-valve, and the persistent period of each temperature is adjustable.The opening and closing of micro-valve is closed and the persistent period is by programme-control.
Integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification provided by the invention, it is easy to integrated with other microfluidic chip technology, it is achieved nucleic acid amplification fully integrated.Such as: nucleic acid extraction unit can be directly integrated, it is achieved nucleic acid purification process and amplification micro-fluidic chip integrally；With the integrated quick detection that can realize drop digital pcr of drop digital pcr.
Integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification provided by the invention, it is possible to achieve multiple condition and detection while multiple sample, can also directly measure with real-time quantitative simultaneously.
Integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification provided by the invention, has an advantage in that:
1, adopting the fluid of fixed temperature to carry out PCR temperature control, temperature fluctuation is little；
2, the temperature controlling instruments that temperature control need not be complicated；
3, temperature switching fast (< 1s), it may be achieved ultrafast nucleic acid amplification；
4, not needing integrated microelectrode, cost is low.
5, this amplification method is prone to integrated with microflow control techniques such as extractions, it is achieved nucleic acid amplification automatization.
Accompanying drawing explanation
The structural representation of the nucleic acid amplification chip of Fig. 1 integrated micro-valve；A () is PCR conversion zone structural representation, (b) is PDMS film structural representation, and (c) is heating fluid channel Rotating fields schematic diagram；D () is gas circuit Rotating fields schematic diagram；E () is the schematic diagram of whole chip；
Wherein: 1PCR conversion zone, 2PDMS thin film, 3 heating fluid channel layers, 4 gas circuit layers, 5 react liquid pool for PCR, and 6 is heating fluid inlet, and 7 for adding the passage of heat, and 8 is heated fluid outlet, and 9 is gas access, and 10 is gas passage, and 11 is the micro-valve of gas,.
Fig. 2 integrated micro-valve nucleic acid amplification chip material object photo；
Fig. 3 integrated micro-valve nucleic acid amplification and drop digital pcr in conjunction with chip photo；
The switching of Fig. 4 integrated micro-valve temperature characterizes；
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 for identification；
Detailed description of the invention
The present invention will be further described by following example, but not thereby limiting the invention.
Before PDMS in the embodiment of the present invention, aggressiveness and cross-linking agent adopt the sylgard184 of Dow Corning Corporation.
A kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, this chip adopts Multi-layer design, mainly includes PCR conversion zone 1, PDMS film layer 2, heating fluid channel layer 3 and gas passage layers 4；Each layer after oxygen plasma processes, key with together with, and be sealed in support glass；
In described PCR conversion zone 1, uniformly 9～100 PCR of arrangement react liquid pool 5；
Described PDMS film layer 2 is surface non-structure, and thickness is the PDMS film of 10～100 microns；
Described heating fluid channel layer 3 includes heating fluid inlet 6, adds the passage of heat 7, heated fluid outlet 8, and adding the passage of heat 7 is roundabout fold-line-shaped, and adding the passage of heat 7 one end has two or three heating fluid inlet 6, and the other end has a heated fluid outlet 7；
Described gas passage layers 4 includes two or 3 gas passages 10, and gas passage 10 one end is 9 gas access 9 other ends is the micro-valve 11 of gas；
PCR conversion zone 1 is the superiors, secondly it is PDMS film layer 2, heating fluid channel layer 3 and gas passage layers 4, the passage of heat 7 that adds of heating fluid channel layer 3 is located immediately at the underface of PCR conversion zone 1PCR reaction tank 5, the micro-valve of gas 11 of gas passage layers 4 and zone of heat liberation layer 3 to add the passage of heat 7 corresponding.
Described chip realizes the switching of nucleic acid amplification each temperature required by integrated micro-valve.
The opening and closing of described micro-valve is closed and the persistent period is by programme-control.
What described chip controlled adds hot fluid is water, air or mineral wet goods.
Described PCR conversion zone can be reaction tank, it is also possible to for reaction channel, its form can design as required.
Embodiment 1
The construction unit of the nucleic acid amplification chip of integrated micro-valve
The preparation method of a kind of integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, the method comprises the steps:
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 aggressiveness before the PDMS of volume ratio 10:1 and cross-linking agent are mixed degassed after, rotary coating is in gas passage template and adds passage of heat template, is placed in 80 DEG C of baking ovens polymerization, is respectively prepared gas passage layers, heating fluid channel layer；
After degassed to aggressiveness before the PDMS of volume ratio 10:1 and cross-linking agent mixing, the mode of cast is adopted to prepare PCR conversion zone；It is made to be polymerized in 80 DEG C of baking ovens；PDMS film layer, directly adopts commercial PDMS film；
The PDMS being polymerized is the superiors according to PCR conversion zone, is secondly PDMS film layer, the order of heating fluid channel layer and gas passage layers, after oxygen plasma processes, key with together with, and be sealed in support glass, complete the preparation of integrated micro-valve micro-fluidic chip.
As it is shown in figure 1, this integrated chip part is broadly divided into PCR reaction zone, PDMS film, heating fluid channel layer and gas passage layers.Whole passage is prepared by PDMS material, wherein the part of the micro-valve of gas passage, when pressure, and upwards deformation, form the pressure to fluid path passage, it is possible to achieve the break-make adding hot fluid of micro-valve.Fluid add between the passage of heat and PCR reaction tank between alternating floor PDMS film, be beneficial to the quick transmission of heat.The steady temperature fluid that chip is adopts water, air or mineral wet goods.Fig. 2 is the photo of simple integrated micro-valve micro-fluidic chip.This chip combined with micro-fluidic other technologies, can also form more complicated micro-fluidic PCR detection chip simultaneously.Fig. 3 is micro-valve integrated chip with numeral drop round pcr in conjunction with micro-fluidic chip, and this integrated chip has more than 10,000 PCR drop fixed cell, it is possible to achieve the quick detection of drop digital pcr.
Embodiment 2
The switching of integrated micro-valve temperature characterizes
In the way of micro-valve control difference is with different dyes fluid, it is possible to this chip temperature is switched acidity and characterizes.Adopt the video recording of CCD digital camera record switching fluid, as shown in Figure 4.This chip can realize the switching of whole chip different dyes fluid in 0.5s, therefore can realize fast PCR reaction.
Embodiment 3
The chip electrophoresis of integrated micro-valve Rapid nucleic acid amplification characterizes and application
Carry out Rapid nucleic acid amplification by this chip, its product is carried out chip electrophoresis separation amplified production is verified.This chip can realize the nucleic acid amplification of 35 circulations in 10 minutes.Chip electrophoresis figure is as shown in Figure 5.This chip also apply be applicable to legal medical expert's identification with electrophoretic separation combination simultaneously, and the STR testing result of actual blood sample is as shown in Figure 6.
Claims (6)
1. the integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification, it is characterised in that this chip adopts Multi-layer design, mainly includes PCR conversion zone, PDMS film layer, heating fluid channel layer and gas passage layers；Each layer after oxygen plasma processes, key with together with, and be sealed in support glass；
In described PCR conversion zone, uniformly 9～100 PCR of arrangement react liquid pool；
Described PDMS film layer is surface non-structure, and thickness is the PDMS film of 10～100 microns；
Described heating fluid channel layer includes heating fluid inlet, adds the passage of heat, heated fluid outlet, and adding the passage of heat is roundabout fold-line-shaped, and heating passage end has two or three heating fluid inlet, and the other end has a heated fluid outlet；
Described gas passage layers includes two or 3 gas passages, and gas passage one end is the gas access other end is the micro-valve of gas；
PCR conversion zone is the superiors, secondly it is PDMS film layer, heating fluid channel layer and gas passage layers, the passage of heat that adds of heating fluid channel layer is located immediately at the underface of PCR conversion zone PCR reaction tank, the micro-valve of gas of gas passage layers and zone of heat liberation layer to add the passage of heat corresponding.
2. the integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification described in claim 1, it is characterised in that this chip realizes the switching of nucleic acid amplification each temperature required by integrated micro-valve.
3. the integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification described in claim 1, it is characterised in that the opening and closing of micro-valve is closed and the persistent period is by programme-control.
4. the integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification described in claim 1, it is characterised in that what this chip controlled adds hot fluid is water, air or mineral wet goods.
5. the micro-fluidic chip of the integrated micro-valve for ultrafast nucleic acid amplification described in claim 1, it is characterised in that PCR conversion zone can be reaction tank, it is also possible to for reaction channel, its form can design as required.
6. the preparation method of the integrated micro-valve micro-fluidic chip for ultrafast nucleic acid amplification described in claim 1, it is characterised in that the method comprises the steps:
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 aggressiveness before the PDMS of volume ratio 10:1 and cross-linking agent are mixed degassed after, rotary coating is in gas passage template and adds passage of heat template, is placed in 80 DEG C of baking ovens polymerization, is respectively prepared gas passage layers, heating fluid channel layer；
After degassed to aggressiveness before the PDMS of volume ratio 10:1 and cross-linking agent mixing, the mode of cast is adopted to prepare PCR conversion zone；It is made to be polymerized in 80 DEG C of baking ovens；PDMS film layer, directly adopts commercial PDMS film；
The PDMS being polymerized is the superiors according to PCR conversion zone, is secondly PDMS film layer, the order of heating fluid channel layer and gas passage layers, after oxygen plasma processes, key with together with, and be sealed in support glass, complete the preparation of integrated micro-valve micro-fluidic chip.
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| CN112322472A (en) * | 2020-11-05 | 2021-02-05 | 上海交通大学 | Instant detection device suitable for nucleic acid detection |
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