CN110923133A - Microfluidic chip and application thereof - Google Patents
Microfluidic chip and application thereof Download PDFInfo
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- CN110923133A CN110923133A CN201811099053.8A CN201811099053A CN110923133A CN 110923133 A CN110923133 A CN 110923133A CN 201811099053 A CN201811099053 A CN 201811099053A CN 110923133 A CN110923133 A CN 110923133A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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Abstract
The invention relates to the technical field of microfluidics, in particular to a microfluidic chip and application thereof. The structural design of the cap-shaped chamber is matched with the control of micro-emulsion droplets (W/O, Water in Oil), each cap-shaped chamber can provide a completely independent reaction chamber, a heating part 6 (such as a heating coil) is arranged at the far opening end or the near opening end of the cap-shaped chamber for temperature circulation, the chip system can amplify thousands of samples simultaneously, and then the change of a fluorescence signal can be directly measured in the cap-shaped chamber by a fluorescent probe to obtain a quantitative result, or the micro-emulsion droplets are pumped out for sequencing and the like. The chip can provide a high-throughput and rapid amplification method and simplify the complicated operation process, so that the polymerase chain reaction can be realized in the microfluidic chip.
Description
Technical Field
The invention relates to the technical field of microfluidics, in particular to a microfluidic chip and application thereof.
Background
The traditional polymerase chain reaction has the defects of complex operation actions of reagents, primer addition and the like in a nucleic acid reaction and a nucleic acid amplification reaction, the amplification process needs to be subjected to multiple times of denaturation, the temperature cycle of jointing and extension, the reaction volume needs to be dozens to hundreds of microliters, the reaction is finished on a 96-well plate, the system is large, and the operation is complex.
Microfluidic chip technology (Microfluidics), also known as Lab-on-a-chip, is capable of integrating the basic functions of conventional biological and chemical laboratories, including sample separation, preparation, chemical reactions, detection, etc., on a few square centimeters microchip.
The micro-fluidic chip has the characteristics of controllable liquid flow, extremely less consumption of samples and reagents, ten-fold or hundred-fold improvement of analysis speed and the like, can simultaneously analyze hundreds of samples in a few minutes or even shorter time, and can realize the whole processes of pretreatment and analysis of the samples on line. Droplet microfluidics is an important branch of microfluidic chip technology. Droplet microfluidic technology was developed over the traditional single-phase microfluidic chip technology, and the three-inlet T-type microfluidic chip design was first proposed by professor runtemf. Compared with a single-phase micro-fluidic system, the system has the advantages of less consumption of samples and reagents, higher mixing speed, difficulty in causing cross contamination, easiness in operation and the like due to the characteristic of water/oil two-phase separation. Therefore, the method has important application in the fields of rapid high-flux detection of pollutants, separation and cultivation of biological samples, observation of chemical reaction progress and the like. The micro-droplets have the advantages of high flux, no cross contamination and the like, and have great application potential in the fields of ink-jet printing, micro-mixing, DNA analysis, material synthesis, protein crystallization and the like.
Therefore, it is of great practical significance to provide a microfluidic chip for nucleic acid amplification.
Disclosure of Invention
In view of this, the present invention provides a microfluidic chip and an application thereof. The microfluidic chip can be used for polymerase chain reaction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a micro-fluidic chip, which comprises a substrate 1, wherein the substrate is provided with a sample inlet 4, a sample outlet 5, a micro-channel 2, a cap-shaped chamber 3, a heating part 6 and an electrode 7;
the cap-shaped cavity 3 is arranged between the sample inlet 4 and the sample outlet 5, and an opening of the cap-shaped cavity 3 is communicated with the micro-channel 2;
the heating part 6 is arranged at the far opening end or the near opening end of the cap-shaped chamber 3;
the electrodes 7 are disposed on a vertical extension line of the cap-shaped chamber 3.
Preferably, the opening length of the longitudinal section of the cap-shaped chamber 3 is not less than the diameter of the target droplet and/or cell.
Preferably, the ratio of the opening length of the longitudinal section of the cap-shaped chamber 3 to the diameter of the target droplet and/or cell is not less than 1.2: 1.
Preferably, the ratio of the opening length of the longitudinal section of the cap-shaped chamber 3 to the diameter of the target droplet and/or cell is (1.2-1.8): 1.
Preferably, the cap-shaped chamber 3 has a rectangular, arched or trapezoidal longitudinal section.
Preferably, the number of the cap-shaped chambers 3 is not less than 1.
The invention also provides application of the microfluidic chip in capturing droplets for nucleic acid amplification.
On the basis, the invention provides a device for capturing liquid drops to perform nucleic acid amplification, which comprises the microfluidic chip.
The invention also provides a method for capturing the liquid drop for nucleic acid amplification, which comprises the following steps:
step 1: obtaining micro-emulsified droplets containing nucleic acid;
step 2: introducing the nucleic acid-containing micro-emulsion droplets, the oil phase and the amplification reagent droplets into the micro-fluidic chip provided by the invention, and adjusting the density of the oil phase to enable the nucleic acid-containing micro-emulsion droplets and/or the amplification reagent droplets to enter the cap-shaped chamber 3;
and step 3: applying an electric field through the electrode 7 to fuse the micro-emulsion droplet containing the nucleic acid and the amplification reagent droplet to obtain a fused droplet;
and 4, step 4: the heating part 6 is turned on, and the fused liquid drops undergo an amplification reaction.
Preferably, the amplification reaction further comprises the step of inverting the microfluidic chip to allow the product droplets to leave the cap-shaped chamber 3, enter the microchannel 2, and be collected.
The invention provides a polymerase chain reaction micro-fluid chip, wherein the structural design of a cap-shaped cavity is matched with the control of micro-emulsion droplets (W/O, Water in Oil), each cap-shaped cavity can provide a completely independent reaction chamber, a heating part 6 (such as a heating coil) is arranged at the far opening end or the near opening end of each cap-shaped cavity for temperature circulation, the chip system can amplify thousands of samples simultaneously, and then the change of a fluorescence signal can be directly measured in the cap-shaped cavity through a fluorescence probe to obtain a quantitative result, or the micro-emulsion droplets are extracted for sequencing and the like. The chip can provide a flux and rapid amplification method and simplify a complicated operation process, so that the polymerase chain reaction can be realized in the microfluidic chip.
The microfluidic chip provided by the invention has the following beneficial effects:
1. the cap-shaped chamber 3 can adjust the length of the longitudinal section opening according to the size of the micro-emulsion drop of the sample and the reagent, so that the micro-emulsion drop of the sample and the reagent only enters the hole singly. If the diameter D of the micro-emulsion droplet is 10-1000 μm, the length and depth of the opening of the cap-shaped chamber 3 along the longitudinal section thereof are within the range
2. Due to the application of the electric field, the surface tension of the micro-emulsion droplets can be changed, and the sample and the reagent micro-emulsion droplets are promoted to be fused for reaction.
3. Because the structure of the micro-fluidic chip and the liquid are extremely micro, the heating part 6 of the heat circulation system can quickly raise and lower the temperature, and accurately control the temperature required by the reaction.
4. The structure of the cap-type chamber 3 can be designed as an array type, so that the microfluidic chip can process several to thousands of samples simultaneously, and the reaction of each sample is performed in a separate reaction chamber without mutual influence.
5. The volume of the sample and the reagent required by the traditional PCR reaction is about 5-50 mu L, and the micro-emulsion droplet technology can reduce the reaction volume from tens of mu L to less than 1nL, so that the volume of the sample and the reagent is greatly reduced (more than ten thousand times saved).
6. The micro-emulsion droplets are controlled by utilizing the density difference of the oil phase and the water phase, so that the micro-emulsion droplets of the product can float out of the cap-shaped cavity 3 through the inversion wafer and are injected into the oil phase, and the micro-emulsion droplets of the product can be simply taken out for subsequent product analysis or sequencing.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Fig. 1 shows a top view and a side view of a microfluidic chip according to the present invention; FIG. 1A is a top view of an array-type cap chamber that can be increased according to experimental requirements; FIG. 1B shows a side view, and the cap-type chamber structure can make micro-emulsion droplets stay in the cap-type chamber due to different specific gravities of water; a heating coil is arranged at the near opening end of the cap-shaped chamber to perform temperature circulation;
FIG. 2 shows the operation flow of the microfluidic chip, and FIG. 2A and FIG. 2B respectively show the sample emulsified droplet and the reagent microemulsified droplet entering the cap-shaped chamber; FIG. 2C shows the application of an additional electric field to promote coalescence of the microemulsified droplets; FIG. 2D illustrates the thermocycling effect; FIG. 2E shows fluorescence detection in place; FIG. 2F shows that the product micro-emulsified droplets are taken out after the reaction and analysis are finished;
wherein, 1-a substrate; 2-micro flow channel; 3-a cap-shaped chamber; 4-sample inlet; 5, a sample outlet; 6-heating means; 7-an electrode; 8-micro-emulsified droplets containing nucleic acids; 9-droplets of amplification reagents; 10-a fused droplet; 11-product droplets.
Detailed Description
The invention discloses a micro-fluidic chip and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The microfluidic chip and the raw materials, components and reagents used in the application of the microfluidic chip provided by the invention can be purchased from the market.
The invention provides a micro-fluidic chip, which comprises a substrate 1, wherein the substrate is provided with a sample inlet 4, a sample outlet 5, a micro-channel 2, a cap-shaped chamber 3, a heating part 6 and an electrode 7;
the cap-shaped cavity 3 is arranged between the sample inlet 4 and the sample outlet 5, and an opening of the cap-shaped cavity 3 is communicated with the micro-channel 2;
the heating part 6 is arranged at the far opening end or the near opening end of the cap-shaped chamber 3;
the electrodes 7 are disposed on a vertical extension line of the cap-shaped chamber 3.
In some embodiments of the invention, the length of the opening of the longitudinal section of the cap-shaped chamber 3 is not less than the diameter of the target droplet and/or cell. Preferably, the ratio of the opening length of the longitudinal section of the cap-shaped chamber 3 to the diameter of the target droplet and/or cell is not less than 1.2: 1. More preferably, the ratio of the opening length of the longitudinal section of the cap-shaped chamber 3 to the diameter of the target droplet and/or cell is (1.2-1.8): 1.
In some embodiments of the invention, the cap chamber 3 has a rectangular, arched or trapezoidal longitudinal section.
In some embodiments of the invention, the number of cap-shaped chambers 3 is not less than 1.
The length of the longitudinal section of the cap-shaped chamber can be changed (1.2< d <1.8) according to the diameter of the analyzed target substance or the diameter of the generated liquid drop, so that the capture of various target substances can be realized. It is easy for those skilled in the art to change the size of the cap-shaped chamber, the shape of the longitudinal section, and the number of the cap-shaped chambers, and the present invention is not described herein, and the length of the longitudinal section of the cap-shaped chamber, the structure and the number of the cap-shaped chambers suitable for the target object are all within the protection scope of the present invention.
The invention also provides application of the microfluidic chip in capturing droplets for nucleic acid amplification.
On the basis, the invention provides a device for capturing liquid drops to perform nucleic acid amplification, which comprises the microfluidic chip.
Obtaining micro-emulsified droplets containing nucleic acid based on the method for carrying out nucleic acid amplification on the captured droplets of the micro-fluidic chip; introducing the nucleic acid-containing micro-emulsion droplets, the oil phase and the amplification reagent droplets into the micro-fluidic chip provided by the invention, and adjusting the density of the oil phase to enable the nucleic acid-containing micro-emulsion droplets and/or the amplification reagent droplets to enter the cap-shaped chamber 3; applying an electric field through the electrode 7 to fuse the micro-emulsion droplets and the amplification reagent droplets to obtain fused droplets; the heating part 6 is turned on, and the fused liquid drops undergo an amplification reaction. And after the amplification reaction is finished, turning over the microfluidic chip to enable the product droplets and the unreacted nucleic acid-containing micro-emulsion droplets and amplification reagent droplets to leave the cap-shaped chamber 3 and enter the micro-channel 2 for collection.
Specifically, the invention combines the micro-emulsification technology, firstly the sample (single cell or DNA), reagent and the like form micro-emulsification liquid drop (W/O), and then the micro-emulsification liquid drop is injected into the micro-fluidic chip (as shown in figure 1) provided by the invention, because the density of the used oil phase is greater than that of the water phase, when the micro-emulsification liquid drop enters the cap-shaped chamber 3, the micro-emulsification liquid drop enters the cap-shaped chamber 3 under the action of buoyancy, and the purpose of isolating the sample micro-emulsification liquid drop is achieved. After the micro-emulsion droplets of the sample enter the holes (see fig. 2A), micro-emulsion droplets of amplification reaction reagents (such as cell lysate, DNA primers, etc.) are injected, and the micro-emulsion droplets of the amplification reaction reagents also enter the cap-shaped chamber 3 (see fig. 2B) due to the buoyancy. Then, an electric field is applied to change the surface tension of the micro-emulsified liquid drop, so that the two liquid drops are fused (as shown in figure 2C). After the droplets are fused, the heating block 6 is turned on to denature, anneal and extend the DNA (FIG. 2D). After the experiment is finished, if the added amplification reaction reagent has a fluorescence signal, the fluorescence intensity can be directly detected in the cap-shaped chamber 3 for analysis (see FIG. 2E). When the product liquid drop is needed to be taken out after the reaction and the analysis are finished, the product liquid drop can be taken out only by turning over the microfluidic chip. The method specifically comprises the following steps: after the micro-fluidic chip is inverted, the product droplets leave the cap-shaped chamber 3 due to the buoyancy, and then oil-phase liquid is injected to enable the product droplets to be output to the micro-fluidic chip and collected for subsequent detection (as shown in fig. 2F).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The microfluidic chip is characterized by comprising a substrate (1), wherein the substrate is provided with a sample inlet (4), a sample outlet (5), a micro-channel (2), a cap-shaped chamber (3), a heating part (6) and an electrode (7);
the cap-shaped cavity (3) is arranged between the sample inlet (4) and the sample outlet (5), and an opening of the cap-shaped cavity (3) is communicated with the micro-channel (2);
the heating part (6) is arranged at the far opening end or the near opening end of the cap-shaped chamber (3);
the electrodes (7) are arranged on a vertical extension line of the cap-shaped chamber (3).
2. The microfluidic chip according to claim 1, wherein the opening length of the longitudinal section of the cap-shaped chamber (3) is not less than the diameter of the target droplet and/or cell.
3. The microfluidic chip according to claim 2, wherein the ratio of the opening length of the longitudinal section of the cap-shaped chamber (3) to the diameter of the target droplet and/or cell is not less than 1.2: 1.
4. The microfluidic chip according to claim 3, wherein the ratio of the opening length of the longitudinal section of the cap-shaped chamber (3) to the diameter of the target droplet and/or cell is (1.2-1.8): 1.
5. Microfluidic chip according to claim 4, characterized in that the cap-shaped chamber (3) has a rectangular, arched or trapezoidal longitudinal section.
6. Microfluidic chip according to claim 5, characterized in that the number of cap-shaped chambers (3) is not less than 1.
7. Use of a microfluidic chip according to any one of claims 1 to 6 for capturing droplets for nucleic acid amplification.
8. An apparatus for capturing droplets for nucleic acid amplification, comprising the microfluidic chip according to any one of claims 1 to 6.
9. A method of capturing droplets for nucleic acid amplification comprising the steps of:
step 1: obtaining micro-emulsified droplets containing nucleic acid;
step 2: introducing the micro-emulsion droplets containing nucleic acid, oil phase and amplification reagent droplets into the micro-fluidic chip according to any one of claims 1 to 6, and adjusting the density of the oil phase to make the micro-emulsion droplets containing nucleic acid and/or the amplification reagent droplets enter the cap-shaped chamber (3);
and step 3: applying an electric field through the electrode (7) to fuse the micro-emulsion droplets and the amplification reagent droplets to obtain fused droplets;
and 4, step 4: and (6) starting the heating component, and carrying out amplification reaction on the fused liquid drops.
10. The method of claim 9, further comprising the step of inverting the microfluidic chip after the amplification reaction has occurred to allow product droplets to leave the cap-shaped chamber (3) and enter the microchannel (2) for collection.
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| CN201811099053.8A CN110923133A (en) | 2018-09-20 | 2018-09-20 | Microfluidic chip and application thereof |
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| CN201811099053.8A CN110923133A (en) | 2018-09-20 | 2018-09-20 | Microfluidic chip and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111778155A (en) * | 2020-07-09 | 2020-10-16 | 墨卓生物科技(上海)有限公司 | PCR amplification mechanism for dPCR integrated micro-fluidic chip |
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2018
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