CN103451088B - Micro-droplet type PCR (polymerase chain reaction) chip and manufacture method thereof - Google Patents
Micro-droplet type PCR (polymerase chain reaction) chip and manufacture method thereof Download PDFInfo
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- CN103451088B CN103451088B CN201310374364.1A CN201310374364A CN103451088B CN 103451088 B CN103451088 B CN 103451088B CN 201310374364 A CN201310374364 A CN 201310374364A CN 103451088 B CN103451088 B CN 103451088B
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Abstract
The invention provides a micro-droplet type PCR (polymerase chain reaction) chip and a manufacture method thereof. The upper layer of the chip is a PDMS (polydimethylsiloxane) imaging cover plate formed by connecting a DNA (deoxyribonucleic acid) target fragment with a primer injection port, a single phase drop forming microstructure, a PCR reaction enzyme and an mRNA (messenger ribonucleic acid) substrate injection port, an oil phase reagent injection port, a drop straight line channel, a cross hybrid channel, a micro drop buffer channel, a micro drop PCR reaction chamber and a micro drop PCR waste liquid chamber, the lower layer of the chip is a glass substrate, and the upper layer and the lower layer are bonded and packaged to form a complete micro drop PCR chip. The manufacture method of the chip comprises the steps of firstly, manufacturing a mask plate; secondly, molding; thirdly, pouring a die; and fourthly, taking a figure. The PDMS figure at the upper layer of the chip adopts a multilayer filter structure, and the formed micro drop has a uniform size, stable grain diameter and strong controllability. Compared with traditional PCR chips, the micro drop PCR chip manufactured by the method has great advantages in hybrid effect, reaction efficiency, and high throughput.
Description
Technical field
The present invention relates to biomedical applications instrument field, particularly, relate to a kind of microlayer model formula pcr chip and preparation method thereof.
Background technology
Polymerase chain reaction (Polymerase Chain Reaction, PCR), or claim acellular clone technology (Free bacteria cloning technique), as molecular biology and an engineered important technology, it is a kind of method of selective amplification DNA and RNA fragment under primer guides, and has the outstanding advantages such as special, responsive, productive rate is high, quick, easy, reproducible, easy automatization.Present stage biotechnology adopt PCR amplification instrument can carry out continuous several times amplification to required goal gene fragment, and the number of the millions of times of orders of magnitude can be copied to, be widely used in by the life science obtained for the purpose of goal gene or gene fragment, engineering in medicine, heredity person who has rendered outstanding service, many fields such as medical diagnosis on disease.
Microlayer model technology develops rapidly at biochemical field, the drop of micro volume can be manipulated based on microlayer model technology, and can realize microlayer model stable generation, surface treatment, break, merge and multilayer drop preparation, therefore microlayer model can be used for the formation etc. that container carries out microorganism culturing, reaction enzymes analysis, microsphere particle.
Find through retrieval, a kind of integrated temperature control PCR-CE micro-fluidic chip and preparation method thereof (China Patent Publication No. 103103120A), describes a kind of structure and making method of normal PCR chip in this patent.
Relative to traditional pcr chip, microlayer model formula pcr chip have output PCR react the particle diameter of drop stablize, homogeneous and can handling strong feature, therefore, it is possible to the fusion of the comparatively conveniently material such as forming reactions enzyme, reactant and catalyzer.
The channel size of micro-fluidic chip is in micron level, and therefore liquid-flow is low reynolds number laminar flow regime.Therefore this flow effect can be utilized to carry out Biochemistry Experiment, but the sensitivity test in microchannel and have oneself shortcoming itself, the mixed effect as the reagent of micro chamber is bad, the demand of sample large, analyzed is low, in mixing process because the rate of flow of fluid difference of hybrid channel causes mixing uneven.And the proposition of microlayer model, mainly for the various problems that reagent mix occurs, utilize two kinds of alternate inconsistent principles, the macromole particle of stable homogeneous will be formed after reagent mix, and alternately carry out PCR reaction at another, make mixing abundant like this, the blocking of hybrid channel can not be caused, make full use of reaction sample reagent, improve the efficiency of the PCR synthesis of reactant.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of microlayer model formula pcr chip and preparation method thereof, produce a kind of with the pcr chip of microlayer model form reaction according to the present invention, more efficiently formation size uniformity, monodispersity and quantity experiment condition have the PCR drop of specific requirement, have and comparatively fast form the features such as a large amount of PCR drop, real-time, cost of manufacture is low.
According to an aspect of the present invention, there is provided a kind of microlayer model formula pcr chip, this chip adopts double layer design, and upper strata is the graphical cover plate of PDMS, lower floor is substrate of glass, and upper and lower two-layer employing vacuum oxygen plasma body bonding packaging is a complete microlayer model pcr chip;
The graphical cover plate of described PDMS from left to right comprises successively: DNA target fragment and primer inlet, single-phase drop molded micro structure, PCR reaction enzymes and mRNA substrate inlet, oil phase reagent inlet, drop beeline channel, cross hybrid channel, microlayer model buffer channel, microlayer model PCR reaction chamber and microlayer model PCR waste chamber, wherein: DNA target fragment and primer inlet, PCR reaction enzymes and mRNA substrate inlet, oil phase reagent inlet are 3 parallel reagent inlets, and each inlet is provided with single-phase drop molded micro structure; Drop beeline channel, cross hybrid channel, drop buffer channel are interconnective reagent mix and circulation passage from left to right; Microlayer model PCR reaction chamber is connected with drop buffer channel, and microlayer model PCR waste chamber is connected with microlayer model PCR reaction chamber.
The present invention at the middle and upper levels graphical cover plate of PDMS adopts negative glue development graphical, and PMDS reverse mould obtains; Oil phase reagent is injected by oil phase reagent inlet, and flow through cross hybrid channel and microlayer model buffer channel by oil phase reagent inlet, enter microlayer model PCR reaction chamber, such oil phase reagent will the whole mixing region of submergence in advance, for the follow-up PCR of obtaining microlayer model creates a continuous environment; Again DNA target fragment and DNA primer and PCR reaction enzymes, mRNA substrate are injected into DNA target fragment and primer inlet, PCR reaction enzymes and mRNA substrate inlet respectively, flow through single-phase drop molded micro structure, utilize the bed filtration characteristic of single-phase drop molded micro structure to be leached by macromolecular substance, macromolecular substance is separated the single-phase drop obtaining stable homogeneous one by one; Drop forms the identical or close material of size by drop beeline channel, mix with the oil phase environment of cross hybrid channel after by drop beeline channel, because the size of cross hybrid channel progressively diminishes, each macromolecular substance that just can be reacted by PCR because of wicking action wraps up one deck oil phase substance, and then forms microenvironment independently microlayer model structure; After microlayer model is stable by microlayer model buffer channel, be stored in microlayer model PCR reaction chamber, and provide microlayer model environment and corresponding reagent material for follow-up PCR reacts.Reagent unnecessary when microlayer model PCR waste chamber is for storing sample introduction, simultaneously as PCR waste liquid relief outlet after completion of the reaction.
Preferably, described single-phase drop molded micro structure adopts one-level filter screen and the decoupling zero of secondary filter, the reagent being about to inject utilizes one-level filter screen that macromolecular substance is carried out single filter, obtain the droplet structure of elementary micro-dimension, and by secondary filter, the droplet structure of elementary micro-dimension is carried out the second-order separation, make its size more microminiaturization, obtain the microlayer model structure of single stable.
More preferably, described one-level filter screen is symmetrical flabellum shape structure; Described secondary filter three row are arranged side by side, and each row is all made up of spacing uniform box-shaped PDMS choker bar, and row are identical with row interval, and choker bar spacing reduces by column.
Preferably, described substrate of glass is as the support of microlayer model pcr chip, and it is provided with alignment symbology.
According to another aspect of the present invention, provide a kind of preparation method of microlayer model pcr chip, the method comprises the steps:
Step one, designed figure is carried out mask processing: copy in one piece of film version or cadmium version by figure by 1:1 size, pending follow-up Mould Machining;
Step 2, employing mold method get figure: use micro-processing technology on one piece of substrate, to process protruding model as mould, peeled away by the graphic material copied and obtain required passageway pattern; Wherein the producing process of mould is as follows: first in clean substrate of glass, sputter one deck Cr/Cu, then last layer Su-8 glue is got rid of on the metal layer, the mask of step one is utilized to carry out photolithography patterning, development, finally in clear water, carry out concussion process, obtain the final mould being carved with this figure; In order to make subsequent process mould clean and tidy, the photoresist material acetone that mould remains and alcohol are successively cleaned concussion.
Step 3, the PDMS prepared watered be filled on mould, the micrographics on mould is copied, utilizes photoresist spinner to be got rid of evenly by PDMS layer, make the figure that takes off smooth, peel off from mould after the solidification of PDMS layer and obtain the cover plate with channel shape.
Step 4, PDMS cover plate become a complete chip with the glass substrate bonding packaging of lower floor: get rid of the PDMS layer that one deck is thin on the glass substrate, dry in baking oven and take out when PDMS becomes semi-solid state, then utilize vacuum oxygen equipment for burning-off photoresist by plasma to peel off substrate to the PDMS cover plate on upper strata and lower floor to remove photoresist, change surface chemistry, after taking-up, two portions are aimed at, are bonded together into complete chip.
Preferably, described aligning is realized by the alignment symbology in the graphical cover plate of PDMS and substrate of glass.
Compared with prior art, the present invention has following beneficial effect:
1, microlayer model pcr chip has relative to traditional pcr chip and can realize fast and the reagent such as reaction enzymes, catalysts rapid fusion, and flux is higher;
2, the present invention considers that the material utilization of PCR process is lower, and early stage utilizes this chip the reactant required for PCR to be created an independently microlayer model environment, thus can not be subject to the impact of external environment in PCR reaction, effectively raise the efficiency of PCR;
3, the PCR microlayer model chip miniaturization made, with low cost, form the microlayer model of PCR reactant easily and efficiently, the DNA synthesis of reacting for follow-up PCR creates superiority condition with being separated; Make use of the MEMS technology in a lot of forward position in this design and fabrication process simultaneously, and later chip to be improved and the innovation of making processes provides very large space.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the graphical cover plate schematic diagram of superstructure PDMS of the present invention;
In Fig. 1: 1 is DNA target fragment and primer inlet, 2 is single-phase drop molded micro structure, 3 is PCR reaction enzymes and mRNA substrate inlet, 4 is oil phase reagent inlet, 5 is drop beeline channel, and 6 is cross hybrid channel, and 7 is drop buffer channel, 8 is microlayer model PCR reaction chamber, and 9 is microlayer model PCR waste chamber;
Fig. 2 is the detail view of single-phase drop molded micro structure of the present invention;
In Fig. 2: 10 is reagent injection port, 11 is one-level filter screen, and 12,13,14 is secondary filter.
Fig. 3 is microlayer model pcr chip overall diagram of the present invention
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
As shown in Figure 3, the present embodiment provides a kind of microlayer model formula pcr chip, and this chip adopts double layer design, and upper strata is the graphical cover plate of PDMS, lower floor is substrate of glass, and upper and lower two-layer employing vacuum oxygen plasma body bonding packaging is a complete microlayer model pcr chip.
As shown in Figure 1 and Figure 2, the graphical cover plate of PDMS of upper strata described in the present embodiment specifically comprises: DNA target fragment and primer inlet 1, single-phase drop molded micro structure 2, PCR reaction enzymes and mRNA substrate inlet 3, oil phase reagent inlet 4, drop beeline channel 5, cross hybrid channel 6, drop buffer channel 7, microlayer model PCR reaction chamber 8 and microlayer model PCR waste chamber 9, and the development of described upper strata PDMS graphical cover plate employing negative glue is graphical, PMDS reverse mould obtains; Wherein: described DNA target fragment and primer inlet 1, PCR reaction enzymes and mRNA substrate inlet 3, oil phase reagent inlet 4 are 3 parallel reagent inlets, and each inlet is provided with single-phase drop molded micro structure 2; Drop beeline channel 5, cross hybrid channel 6, drop buffer channel 7 are interconnective reagent mix and circulation passage from left to right; Microlayer model PCR reaction chamber 8 is connected with drop buffer channel 7, and microlayer model PCR waste chamber 9 is connected with microlayer model PCR reaction chamber 8.
In the present embodiment, oil phase reagent is injected by described oil phase reagent inlet 4, and oil phase reagent is flow through cross hybrid channel 6 and buffer channel 7 successively by oil phase reagent inlet 4, enter in microlayer model PCR reaction chamber 8, such oil phase reagent will the whole mixing region of submergence in advance, for the follow-up PCR of obtaining microlayer model creates a continuous environment; Again DNA target fragment and DNA primer and PCR reaction enzymes, substrate are injected into DNA target fragment and primer inlet 1, PCR reaction enzymes and mRNA substrate inlet 3 respectively, single-phase drop molded micro structure 2 is after drop injects, utilize one-level filter screen 11 and secondary filter 12,13,14 to be leached by macromolecular substance, macromolecular substance is by the single-phase droplet structure obtaining stable homogeneous after a separation; Single-phase drop just forms size similar substance by drop beeline channel 5; After by drop beeline channel 5, just mix with the oil phase environment of cross hybrid channel 6, because the size of cross hybrid channel 6 progressively diminishes, each macromolecular substance that just can be reacted by PCR because of wicking action wraps up one deck oil phase substance, and then forms microenvironment independently microlayer model structure; After microlayer model is stable by drop buffer channel 7, is stored in microlayer model PCR reaction chamber 8, and provides microlayer model environment and corresponding reagent material for follow-up PCR reacts.
As shown in Figure 2, in the present embodiment, single-phase drop molded micro structure 2 adopts one-level filter screen 11 and secondary filter 12, 13, 14 decoupling zeros, wherein: one-level filter screen 11 flabellum shape structure symmetrically, secondary filter 12, 13, 14 3 row are arranged side by side, first macromolecular substance is carried out single filter through one-level filter screen 11 by the reagent injected by reagent injection port 10, obtain the droplet structure of elementary micro-dimension, then by three row secondary filters 12 side by side, 13, the droplet structure of elementary micro-dimension is carried out the second-order separation by 14, make its size more microminiaturization, obtain single stable, the microlayer model structure that size is suitable, each row secondary filter 12,13,14 is all be made up of the uniform choker bar of spacing, and the spacing of choker bar reduces by column.Choker bar in filter screen and filter is formed by PDMS, has good biocompatibility.
Preparation method's step of a kind of microlayer model pcr chip described in the present embodiment comprises:
Step one, designed figure is carried out mask processing: copy in one piece of film version by figure by 1:1 size, pending follow-up Mould Machining;
Step 2, employing mold method get figure: use micro-processing technology on one piece of substrate, to process protruding model as mould, peeled away by the graphic material copied and obtain required passageway pattern; Wherein the producing process of mould is as follows: first in clean substrate of glass, sputter one deck Cr/Cu, then last layer Su-8 glue is got rid of on the metal layer, the mask of step one is utilized to carry out photolithography patterning, development, finally in clear water, carry out concussion process, obtain the final mould being carved with this figure; In order to make subsequent process mould clean and tidy, the photoresist material acetone that mould remains and alcohol are successively cleaned concussion;
Step 3, the PDMS prepared watered be filled on mould, the micrographics on mould is copied, utilizes photoresist spinner to be got rid of evenly by PDMS layer, make the figure that takes off smooth, peel off from mould after the solidification of PDMS layer and obtain the cover plate with channel shape;
Step 4, PDMS cover plate become a complete chip with the glass substrate bonding packaging of lower floor: get rid of the PDMS layer that one deck is thin on the glass substrate, dry in baking oven and take out when PDMS becomes semi-solid state, then utilize vacuum oxygen equipment for burning-off photoresist by plasma to peel off substrate to the PDMS cover plate on upper strata and lower floor to remove photoresist, change surface chemistry, after taking-up, two portions are aimed at, are bonded together into complete chip.
Produce a kind of with the pcr chip of microlayer model form reaction according to the present invention, more efficiently formation size uniformity, monodispersity and quantity experiment condition have the PCR drop of specific requirement, have and comparatively fast form the features such as a large amount of PCR drop, real-time, cost of manufacture is low.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (5)
1. a microlayer model formula pcr chip, is characterized in that, this chip adopts double layer design, and upper strata is the graphical cover plate of PDMS, and lower floor is substrate of glass, and upper and lower two-layer employing vacuum oxygen plasma body bonding packaging is a complete microlayer model pcr chip;
The graphical cover plate of described PDMS specifically comprises: DNA target fragment and primer inlet, single-phase drop molded micro structure, PCR reaction enzymes and mRNA substrate inlet, oil phase reagent inlet, drop beeline channel, cross hybrid channel, microlayer model buffer channel, microlayer model PCR reaction chamber and microlayer model PCR waste chamber, and the development of described PDMS graphical cover plate employing negative glue is graphical, PMDS reverse mould obtains; Wherein: DNA target fragment and primer inlet, PCR reaction enzymes and mRNA substrate inlet, oil phase reagent inlet are 3 parallel reagent inlets, and each inlet is provided with single-phase drop molded micro structure; Drop beeline channel, cross hybrid channel, drop buffer channel are interconnective reagent mix and circulation passage from left to right; Microlayer model PCR reaction chamber is connected with drop buffer channel, and microlayer model PCR waste chamber is connected with microlayer model PCR reaction chamber; Oil phase reagent is injected by oil phase reagent inlet, and flow through cross hybrid channel and microlayer model buffer channel by oil phase reagent inlet, enter microlayer model PCR reaction chamber, such oil phase reagent will the whole mixing region of submergence in advance, for the follow-up PCR of obtaining microlayer model creates a continuous environment; Again DNA target fragment and DNA primer and PCR reaction enzymes, mRNA substrate are injected into DNA target fragment and primer inlet, PCR reaction enzymes and mRNA substrate inlet respectively, flow through single-phase drop molded micro structure, utilize the bed filtration characteristic of single-phase drop molded micro structure to be leached by macromolecular substance, macromolecular substance is separated the single-phase drop obtaining stable homogeneous one by one; Drop forms the identical or close material of size by drop beeline channel, mix with the oil phase environment of cross hybrid channel after by drop beeline channel, the size of cross hybrid channel progressively diminishes, each macromolecular substance providing wicking action to be reacted by PCR wraps up one deck oil phase substance, and then forms microenvironment independently microlayer model structure; After microlayer model is stable by microlayer model buffer channel, be stored in microlayer model PCR reaction chamber, and provide microlayer model environment and corresponding reagent material for follow-up PCR reacts;
Described single-phase drop molded micro structure adopts one-level filter screen and the decoupling zero of secondary filter, and described one-level filter screen is symmetrical flabellum shape structure; Described secondary filter three row are arranged side by side, and each row is all made up of spacing uniform box-shaped PDMS choker bar, and row are identical with row interval, and choker bar spacing reduces by column.
2. a kind of microlayer model formula pcr chip according to claim 1, it is characterized in that, described single-phase drop molded micro structure adopts one-level filter screen and the decoupling zero of secondary filter, the reagent being about to inject utilizes one-level filter screen that macromolecular substance is carried out single filter, obtain the droplet structure of elementary micro-dimension, and by secondary filter, the droplet structure of elementary micro-dimension is carried out the second-order separation, make its size more microminiaturization, obtain the microlayer model structure of single stable.
3. a kind of microlayer model formula pcr chip according to any one of claim 1-2, it is characterized in that, described substrate of glass is as the support of microlayer model pcr chip, and it is provided with alignment symbology.
4. a preparation method for microlayer model pcr chip according to claim 1, it is characterized in that, the method comprises the steps:
Step one, designed figure is carried out mask processing: copy in one piece of film version or cadmium version by figure by 1:1 size, pending follow-up Mould Machining;
Step 2, employing mold method get figure: use micro-processing technology on one piece of substrate, to process protruding model as mould, peeled away by the graphic material copied and obtain required passageway pattern; Wherein the producing process of mould is as follows: first in clean substrate of glass, sputter one deck Cr/Cu, then last layer Su-8 glue is got rid of on the metal layer, the mask of step one is utilized to carry out photolithography patterning, development, finally in clear water, carry out concussion process, obtain the final mould being carved with this figure; The photoresist material acetone that mould remains and alcohol are successively cleaned concussion;
Step 3, the PDMS prepared watered be filled on mould, the micrographics on mould is copied, utilizes photoresist spinner to be got rid of evenly by PDMS layer, make the figure that takes off smooth, peel off from mould after the solidification of PDMS layer and obtain the cover plate with channel shape;
Step 4, PDMS cover plate become a complete chip with the glass substrate bonding packaging of lower floor: get rid of the PDMS layer that one deck is thin on the glass substrate, dry in baking oven and take out when PDMS becomes semi-solid state, then utilize vacuum oxygen equipment for burning-off photoresist by plasma to peel off substrate to the PDMS cover plate on upper strata and lower floor to remove photoresist, change surface chemistry, after taking-up, two portions are aimed at, are bonded together into complete chip.
5. the preparation method of a kind of microlayer model pcr chip according to claim 4, it is characterized in that, described substrate of glass is as the support of microlayer model pcr chip, and it is provided with alignment symbology; Described aligning is realized by the alignment symbology in the graphical cover plate of PDMS and substrate of glass.
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| CN104109628B (en) * | 2014-07-17 | 2016-04-13 | 重庆大学 | Based on the drop formula PCR reactor of LASER HEATING |
| CN104593256B (en) * | 2015-01-06 | 2016-08-31 | 上海交通大学 | The reusable pcr chip of electrode |
| WO2017061620A1 (en) * | 2015-10-09 | 2017-04-13 | シスメックス株式会社 | Analyte treatment chip, analyte treatment device, and analyte treatment method |
| CN205874440U (en) * | 2016-04-08 | 2017-01-11 | 周辉 | Ribonucleic chain polymerization amplification reaction detection device |
| CN108148744A (en) * | 2016-12-06 | 2018-06-12 | 中山百慧生物科技有限公司 | A kind of drop number pcr chip and corresponding method of detection and detecting system |
| CN106701556B (en) * | 2017-01-06 | 2019-02-12 | 广东顺德永诺生物科技有限公司 | Digital pcr detection device and its liquid channel system |
| CN107090406B (en) * | 2017-03-15 | 2019-07-16 | 深圳先进技术研究院 | micro-droplet PCR chip and manufacturing method thereof |
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| CN107433213B (en) * | 2017-07-11 | 2020-01-03 | 东南大学 | Three-dimensional parallel type multiple emulsion rapid preparation device |
| EP3674393A4 (en) * | 2017-11-06 | 2020-11-11 | Targetingone Corporation | Droplet detection apparatus |
| CN109746061A (en) * | 2017-11-06 | 2019-05-14 | 北京新羿生物科技有限公司 | Microlayer model generating means |
| CN109112063A (en) * | 2018-08-20 | 2019-01-01 | 北京旌微医学工程研究院有限公司 | A kind of detection of nucleic acids micro-fluidic chip and preparation method thereof |
| CN109182092A (en) * | 2018-10-19 | 2019-01-11 | 苏州德思普生物科技有限公司 | A kind of micro-fluidic chip and its application for detection of nucleic acids |
| CN110452815A (en) * | 2019-08-16 | 2019-11-15 | 苏州译酶生物科技有限公司 | A kind of archaeal dna polymerase continuously-directional evolution bacterial strain screening chip and its method |
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