WO2021043224A1 - 一种在微流控芯片上生成液滴阵列的方法和装置 - Google Patents

一种在微流控芯片上生成液滴阵列的方法和装置 Download PDF

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Publication number
WO2021043224A1
WO2021043224A1 PCT/CN2020/113313 CN2020113313W WO2021043224A1 WO 2021043224 A1 WO2021043224 A1 WO 2021043224A1 CN 2020113313 W CN2020113313 W CN 2020113313W WO 2021043224 A1 WO2021043224 A1 WO 2021043224A1
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chip
array
lower chip
upper chip
microfluidic
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PCT/CN2020/113313
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English (en)
French (fr)
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沈峰
屈海军
吕蔚元
迂妍
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上海交通大学
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Priority to US17/635,218 priority Critical patent/US20220220548A1/en
Publication of WO2021043224A1 publication Critical patent/WO2021043224A1/zh

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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
    • B01L3/502738—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 characterised by integrated valves
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0605—Metering of fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • 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
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • 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
    • B01L2300/0887—Laminated structure
    • 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
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/065—Valves, specific forms thereof with moving parts sliding valves

Definitions

  • the invention relates to the field of droplet generation, in particular to a method and device for generating a droplet array on a microfluidic chip.
  • Droplets are widely used in physics, chemistry, biology and medicine. Among them, a large number (usually more than 100) droplet arrays show unique advantages in gene, protein and cell analysis.
  • each microdroplet or microwell contains at most one copy of the target gene.
  • some microdroplets or microwells contain a target gene, and other microdroplets or microwells do not contain the target gene.
  • a detectable signal such as a fluorescent signal.
  • the quantitative methods of digital PCR are mainly divided into the flow-generating micro-droplet method and the chip method based on reaction micro-holes. Both of these methods achieve the purpose of digital PCR by dispersing the reaction solution into a large number of micro-reaction units.
  • the flow-generating droplet method is mainly through the design of a special microfluidic fluid channel, using organic liquid to cut off the aqueous solution to generate a series of droplets.
  • this method please refer to Angew.Chem.Int.Ed.2006,45,7336-7356.
  • One of the representatives of the fluid method is cross-flowing droplet formation. In this method, the organic phase and the water phase flow at an angle (T-shaped or Y-shaped) to each other, and the water phase is stretched by shear force and finally liquid droplets are generated.
  • Another representative method is flow focusing droplet formation. This method generates droplets through a confined narrow area through the non-parallel flow of the organic term and the water phase.
  • Co-flowing droplet formation Another method is co-flowing droplet formation (Co-flowing droplet formation), this method is to make the dispersed phase (for example, water phase) channel is enclosed in the continuous phase (for example, organic phase) channel, in the dispersed phase channel At the end, the fluid is stretched until the shear force breaks it to form droplets.
  • ddPCR BioRad's droplet digital PCR system
  • Bó Lé’s droplet digital PCR system is equipped with a set of droplet generation chip, which can generate tens of thousands of nano-upgradable micro droplets relatively quickly.
  • the micro-droplets are amplified in a thermal cycler, and the fluorescence of the micro-droplets is detected by a liquid fluorescence detection system similar to flow detection.
  • the chip method mainly forms micropores or microreaction cells on a microfluidic chip, and then disperses the aqueous solution of the dispersed phase into the micropores or microreaction cells, so that the water phase in the micropores forms relatively independent microdroplets.
  • the most representative one is Thermo's QuantStudio 3D digital PCR system.
  • Thermo Fisher's system has a microfluidic chip containing tens of thousands of reaction micropores. The reaction solution is dispersed into these micropores, and then these micropores are covered with an organic phase (oil phase) to form independent reaction micropores.
  • a SlipChip is a microfluidic chip with a large number of micropores prepared on the lower surface of the upper chip and the upper surface of the lower chip.
  • the upper and lower chiplets are assembled together, and the micropores of the upper and lower chiplets are partially superimposed to form a connected fluid channel.
  • the upper and lower sub-chips slide relatively, and the micropores no longer partially overlap each other, thereby generating a large number of droplets.
  • This method requires precise alignment of a large number of micro-holes of the upper sub-chip and a large number of micro-holes of the lower sub-chip at the initial position to ensure that the droplets are smoothly added to the micro-holes.
  • the disadvantages of the prior art mainly include the following points:
  • the droplet method requires precise control of the flow rates of two immiscible liquids. This process is usually Requires the aid of fluid pumps and other instruments, the overall instrument system will be more complicated, the instrument volume is large, the system is more expensive, and the uniformity of the droplets is very important to the accuracy and reliability of the analysis results such as digital PCR.
  • the uniformity of the droplets is very important to the accuracy and reliability of the analysis results such as digital PCR.
  • surfactant surfactant
  • the QuantStudio 3D digital PCR system requires a lot of manual operation steps, and the process of generating droplets is more complicated, and it divides the aqueous solution in the micropores through the organic phase (oil phase), which is prone to cross-contamination between the micropores.
  • the micro-valve-controlled microfluidic chip studied by Stephen Quake also requires a complex pressure control system (to control the microvalve), and the processing cost of the chip (consumables) is very high; the method of SlipChip needs to be improved.
  • the large number of micro-holes of the daughter chip and the large number of micro-holes of the lower daughter chip are accurately aligned at the initial position to ensure that the droplets are smoothly added to the micro-holes, and the requirements for chip processing, assembly and control are high.
  • the droplet arrays can be effectively and controllably formed through a simple combination of upper and lower chips and simple operation methods. It is simple, effective and sufficient to avoid cross-contamination through physical isolation.
  • the technical problem to be solved by the present invention is how to provide a method for generating a droplet array on a microfluidic chip, which can be effective through a simple upper and lower chip combination device and simple operation method
  • the droplet array can be formed in a controllable manner, and the cross-contamination phenomenon can be avoided simply, effectively and fully through physical isolation, which overcomes the shortcomings of the prior art.
  • a method for generating a droplet array on a microfluidic chip which includes the following steps:
  • Step 1 Assemble the upper chip and the lower chip to the initial position, the fluid pipe of the upper chip and the micropore array of the lower chip partially or completely cover; the fluid pipe of the upper chip contains one or more connected fluids The structure of the channel;
  • Step 2 Inject the solution into the chip, and the solution partially or completely fills the microwell array of the lower chip;
  • Step 3 The upper chip and the lower chip are relatively moved to the liquid separation position, the fluid pipe of the upper chip and the microwell array of the lower chip no longer overlap, and the solution is dispersed in the microwell array In, an array of droplets is formed.
  • the microfluidic chip includes the upper chip and the lower chip, wherein the lower surface of the upper chip and the upper surface of the lower chip are in contact with each other, and the lower surface of the upper chip is in contact with each other
  • the upper surface of the lower chip and the upper surface need to be subjected to a hydrophobic modification treatment; the upper chip or the lower chip is provided with liquid inlet holes, and the upper chip or the lower chip may also be provided with liquid outlet holes.
  • the nature of the fluid channel of the upper chip can be linear, curved, or a combination design of the two.
  • the size specification range of the fluid channel of the upper chip is: a width of 1 ⁇ m-10 cm, a length of 100 ⁇ m-100 cm, and a depth of 1 ⁇ m-1 cm.
  • the surface of the fluid channel of the upper chip needs to be hydrophobicized or hydrophilized.
  • the micropore array of the lower chip may include one or more micropores, and the size and depth of the micropores can be designed to be consistent or different; the surface of the micropores needs to be subjected to surface modification treatment, so The surface modification treatment can be selected from one or more of physical modification, chemical modification, and biological modification.
  • an organic phase may be injected into the chip first, and the organic phase may include a surface chemical composition that is hydrophobically modified.
  • the material of the upper chip and the lower chip may be selected from any one of glass, quartz, plastic, ceramic, and paper materials.
  • the upper chip and the lower chip can be prepared by photolithography, hydrofluoric acid wet etching, dry etching, and hot embossing.
  • one or more expansion channels may be designed on the upper chip, and the expansion channels may be filled with air or organic phase, and when the upper chip and the lower chip move to the liquid dividing position relatively , The expansion channel overlaps the micro-hole array of the lower chip.
  • a microfluidic chip for generating a droplet array comprising:
  • the upper chip, the fluid pipe of the upper chip is a structure including one or more connected fluid channels;
  • the lower chip, the lower chip is provided with a micro-hole array
  • the fluid pipe of the upper chip covers part or all of the microwell array of the lower chip
  • the solution when the solution is injected into the chip, the solution partially or completely fills the microwell array of the lower chip; and then the upper chip and the lower chip are relatively moved to the liquid separation position, and the fluid of the upper chip The pipe and the microwell array of the lower chip no longer overlap, so that the solution is dispersed into the microwell array to form a droplet array.
  • the lower surface of the upper chip and the upper surface of the lower chip are in contact with each other.
  • the lower surface of the upper chip and the upper surface of the lower chip that are in contact with each other are treated by hydrophobic modification.
  • the upper chip or the lower chip is provided with a liquid inlet hole, and the upper chip or the lower chip may also be provided with a liquid outlet hole.
  • the nature of the fluid channel of the upper chip is linear, curvilinear or a combination design of the two.
  • the size range of the fluid channel of the upper chip is as follows: width is 1 ⁇ m-10 cm, length is 100 ⁇ m-100 cm, and depth is 1 ⁇ m-1 cm.
  • the surface of the fluid channel of the upper chip is modified by hydrophobization or hydrophilization.
  • the microwell array of the lower chip may include a plurality of microwells.
  • the surface of the micropores is surface-modified.
  • the surface modification treatment can be selected from one or more of physical modification, chemical modification, and biological modification.
  • the materials of the upper chip and the lower chip are selected from the group consisting of glass, quartz, plastic, ceramic, paper material, or a combination thereof.
  • the materials of the upper chip and the lower chip are selected from the group consisting of glass, quartz, plastic, ceramic, or a combination thereof.
  • the upper chip and the lower chip are prepared by photolithography, hydrofluoric acid wet etching, dry etching, hot embossing, injection molding, and 3D printing.
  • one or more expansion channels are provided on the upper chip, and air or organic phase is filled in the expansion channels, and when the upper chip and the lower chip move relatively to the liquid separation In the position, the expansion channel overlaps the micro-hole array of the lower chip.
  • the density of micropores is 4-100,000 holes/cm 2 , preferably 9-9000 holes/cm 2 , more preferably 25-5000 holes/cm 2 or 100 -5000 holes/cm 2 .
  • the volume of each micropore is 0.001-100 nanoliters, preferably 0.01-50 nanoliters, more preferably 0.05-10 nanoliters, and most preferably 0.1-5 nanoliters.
  • the ratio (D/S 1/2 ) of the depth D of each micropore to the cross-sectional area S 1/2 (D/S 1/2) is 1/200 to 1, preferably 1/20-0.8, more preferably Land 1/5-0.5.
  • the ratio (D/A) of the depth D of each micropore to the length A of the square is 1/200 to 1, preferably 1/20-0.8 , Better 1/5-0.5.
  • the depth D of the micropores is 5-200 micrometers, preferably 10-100 micrometers, more preferably 20-50 micrometers.
  • the ratio (D/d) of the depth D of each micropore to the length d of the circular shape is 1/200 to 1, preferably 1/20 -0.8, more preferably 1/5-0.5.
  • the present invention has at least the following beneficial technical effects:
  • the present invention does not need to overlap the micro-holes of the upper chip and the lower chip to establish a connected fluid channel, which is simpler in chip processing and can allow larger tolerances. In terms of chip assembly, precise alignment operations are no longer required, and assembly is more convenient;
  • the present invention does not require a complicated control system, and can effectively control the size and shape of the generated droplets;
  • the present invention can perform better physical isolation, so that there is no cross-contamination between the microwells.
  • FIG. 1 is a combined diagram of the positions of the upper and lower chips after moving in a preferred embodiment of the present invention
  • Figure 2 is a combined view of the initial position of the upper and lower chips of a preferred embodiment of the present invention
  • Figure 3 is a bottom view of the upper chip of a preferred embodiment of the present invention.
  • Figure 4 is a top view of a lower chip of a preferred embodiment of the present invention.
  • Fig. 5 is a graph of fluorescence signals of three adjacent microwells before digital PCR amplification in a preferred embodiment of the present invention
  • Fig. 6 is a graph of fluorescence signals of three adjacent microwells after digital PCR amplification in a preferred embodiment of the present invention
  • FIG. 7 is a schematic diagram of an upper chip with an expansion channel according to a preferred embodiment of the present invention.
  • FIG. 8 is a combined diagram of the initial positions of the upper chip and the lower chip with an expansion channel according to a preferred embodiment of the present invention.
  • FIG. 9 is a combined diagram of the upper chip and the lower chip with an expansion channel after moving in a preferred embodiment of the present invention.
  • FIG. 10 is a top view of the lower chip of another preferred embodiment of the present invention, in which micropores of different sizes are provided;
  • FIG. 11 is a combined diagram of the initial positions of the upper chip and the lower chip with expansion channels according to another preferred embodiment of the present invention.
  • FIG. 12 is a combined diagram of the positions of the upper chip and the lower chip with an expansion channel after moving according to another preferred embodiment of the present invention.
  • FIG. 13 is a top view of a lower chip of another preferred embodiment of the present invention, in which micro holes of different shapes are provided.
  • FIG. 14 is a combined diagram of the upper chip with expansion channels and the lower chip shown in FIG. 13 in an initial position;
  • FIG. 15 is a combined diagram of the upper chip with the expansion channel and the lower chip shown in FIG. 13 after being moved in a liquid dividing position;
  • Fig. 16 shows a schematic diagram of the lower chip of another preferred embodiment of the present invention, in which different microstructures can be provided in a micro unit.
  • FIG. 17 is a reaction process after moving the upper chip and the lower chip with extended channels in digital PCR
  • Fig. 18 shows another preferred embodiment of the present invention to generate a uniform-sized microwell array.
  • Figure 19 shows the nucleic acid quantitative detection results obtained by using the digital PCR system based on the microfluidic chip of the present invention.
  • the displacement microfluidic chip of the present invention can quickly, efficiently and simply disperse the solution injected into the chip (such as the reaction solution of digital PCR) through the relative sliding of the upper chip and the lower chip, that is, when sliding from the initial position to the liquid dividing position In the microwell array of the lower chip, an array of droplets is formed.
  • the present invention has been completed on this basis.
  • chip-on As used herein, the terms “chip-on” and “chip-on board” are used interchangeably.
  • lower chip As used herein, the terms “lower chip” and “lower chip board” are used interchangeably.
  • upper, lower, lower, left, and “right” are relative, and they are used to express relative spatial position relationships.
  • the upper chip may also be called the lower chip, and the lower chip may also be called the upper chip.
  • the term "between”, when used in the context of movement between the "first position” and the “second position”, can refer to movement only from the first position to the second position, only from The second position moves to the first position, or moves from the first position to the second position and from the second position to the first position.
  • the first position is the initial position where the upper chip and the lower chip are assembled, or the injection position of the upper chip and the lower chip when a fluid (such as a solution) is injected into the chip; the second position is the fluid dividing position.
  • the terms "chip of the present invention”, “microfluidic chip of the present invention”, “displacement microfluidic chip”, and “displacement microfluidic chip of the present invention” are used interchangeably and refer to the first aspect of the present invention.
  • the chip can disperse the solution injected into the chip into the microwell array of the lower chip when the chip slides from the initial position to the liquid separation position through the relative sliding of the upper chip and the lower chip , Form an array of droplets.
  • the displacement microfluidic chip of the present invention includes an "upper chip” and a “lower chip” that are used in cooperation with each other.
  • the upper chip includes a structure of one or more connected fluid channels, and the size range of the fluid channels is as follows: width is 1 ⁇ m-10cm, length is 100 ⁇ m-100cm, and depth is 1 ⁇ m-1cm.
  • the microfluidic chip of the present invention can be used to generate droplet arrays of different sizes and shapes.
  • the upper chip is provided with a liquid inlet hole.
  • the upper chip may also be provided with liquid outlet holes.
  • one or more expansion channels are provided on the upper chip, and the expansion channels are filled with air or organic phase.
  • the solution in the upper expansion channel of the chip can be used as an additional reaction solution to increase the overall reaction solution volume, thereby achieving the purpose of improving reaction sensitivity.
  • the lower chip is provided with a micro-hole array.
  • the micropore density is not particularly limited. Typically, the micropore density is 4-100,000 pores/cm 2 , preferably 9-9000 pores/cm 2 , more preferably 25-5000 pores/cm 2 or 100-5000 pores/cm 2 .
  • the size and depth of the micropores can be designed to be consistent or different.
  • the lower chip may contain wells of different sizes to generate liquid units with different volumes.
  • the lower chip may also include micropores with different depths to generate liquid cells with different depths.
  • the lower chip may also include dimples of different shapes.
  • Representative shapes include (but are not limited to): circle, rectangle, square, cross, triangle, or other arbitrary shapes.
  • the surface of the micropores may be unsurface-treated or surface-modified.
  • Representative surface modification treatments include (but are not limited to): physical modification, chemical modification, biological modification, or a combination thereof.
  • the surface of the chip of the present invention is modified using a gas phase silanization method, for example, dimethyldichlorosilane is used to perform a hydrophobic modification treatment on the glass surface.
  • a gas phase silanization method for example, dimethyldichlorosilane is used to perform a hydrophobic modification treatment on the glass surface.
  • the invention also provides a method for generating a droplet array based on the displacement microfluidic chip of the invention.
  • the method includes:
  • Step 1 Provide the displacement microfluidic chip of the present invention, wherein the upper chip and the lower chip are in initial positions; at this time, the fluid pipe of the upper chip partially or completely covers the microwell array of the lower chip;
  • Step 2 Inject the solution into the chip, so that the solution partially or completely fills the microwell array of the lower chip;
  • Step 3 Relatively move (or slide) the upper chip and the lower chip to the liquid separation position. At this time, the fluid pipes of the upper chip and the micro-hole array of the lower chip no longer overlap, so that all The solution is dispersed into the microwell array to form an array of droplets.
  • the displacement microfluidic chip of the present invention is provided with an expansion channel, and the expansion channel contains organic phase components
  • the representative organic phase is a mixture of mineral oil and tetradecane, for example, a mixture formed by mixing in equal volumes.
  • a layer of organic phase liquid is added between the upper and lower chips and combined in the initial sample loading position, the connected fluid channels of the upper chip and the micropores of the lower chip are occupied by the organic phase.
  • the invention also provides applications of the displacement microfluidic chip of the invention and the generation of droplet arrays.
  • microfluidic chip of the present invention through simple operations on the upper chip and the lower chip, an array containing a large number of micro-droplets (such as 1000-10000 or more micro-droplets) can be effectively and controllably formed.
  • the device and method of the present invention can be applied to applications that require a large amount of independent micro-liquid.
  • a typical application is to use the displacement microfluidic chip of the present invention in a digital PCR reaction to quantitatively detect nucleic acid samples.
  • the micro-droplet array can be effectively and controllably formed by a simple device of upper and lower chip combination and a simple operation method, and the cross-contamination phenomenon can be avoided simply, effectively and sufficiently by means of physical isolation.
  • the present invention does not need to overlap the micropores of the upper chip and the lower chip to establish a connected fluid channel, which is simpler in chip processing, can allow larger tolerances, and no longer requires precision in chip assembly.
  • the alignment operation makes assembly more convenient.
  • the present invention does not require a complicated control system, and can effectively control the size and shape of the generated droplets.
  • an upper chip (as shown in FIG. 3) and a lower chip (as shown in FIG. 4) are prepared on the glass material by a wet etching method.
  • the fluid channel of the upper chip has a width of 5 mm, a length of 15 mm, and a depth of 50 microns.
  • the upper chip contains a liquid inlet and a liquid outlet.
  • the schematic diagram of the structure of the microfluidic chip is shown in Figs. 3 and 4.
  • the actually prepared lower chip contains 5000 micropores distributed in an area 4.5 mm wide and 12.5 long.
  • the microholes of the lower chip are etched with a diameter of 80 microns and a depth of 25 microns.
  • the surface of the chip uses a vapor phase silanization method, and dimethyldichlorosilane is used to perform a hydrophobic modification treatment on the glass surface.
  • the organic phase consists of mineral oil and tetradecane mixed in equal volumes.
  • a layer of organic phase liquid is added between the upper and lower chips, and the relative positions of the two are placed in the initial sample loading position as shown in FIG.
  • the fluid channels and the micropores of the lower chip are occupied by the organic phase.
  • an aqueous solution containing polyethylene glycol octyl phenyl ether and fluorescein is injected into the chip.
  • the aqueous solution will replace the organic phase in the fluid channels and micropores.
  • the fluid channel of the upper chip and the microwell array of the lower chip are staggered to the liquid separation position as shown in FIG. 1, and the liquid in the microwell array of the lower chip forms a microdroplet and microwell array.
  • the chip preparation is the same as in Example 1.
  • the upper chip and the lower chip are prepared on the glass material by wet etching.
  • the fluid channel of the upper chip has a width of 5 mm, a length of 15 mm, and a depth of 50 microns.
  • the upper chip contains a liquid inlet and a liquid outlet.
  • the lower chip contains 5000 micropores distributed in an area of 4.5 mm wide and 12.5 long.
  • the microholes of the lower chip are etched with a diameter of 80 microns and a depth of 25 microns.
  • the surface of the chip uses a vapor phase silanization method, and dimethyldichlorosilane is used to perform a hydrophobic modification treatment on the glass surface.
  • the organic phase consists of mineral oil and tetradecane mixed in equal volumes. A layer of organic phase liquid is added between the upper and lower chips. When combined in the initial position of sample application, the connected fluid channels of the upper chip and the micropores of the lower chip are occupied by the organic phase.
  • PCR reaction solution preparation 50 microliters of reaction reagents include: 1.25 microliters of primer-1: CAGCGAGTCAGTGAGCGAGGAA (SEQ ID No:1); 1.25 microliters of primer-2: TGTAAAGCCTGGGGTGCCTAA (SEQ ID No: 2); 25 microliters of EvaGreen 2x PCR reaction solution (purchased from Bó Lé); 15 microliters of PCR reagent water; 2.5 microliters of 10 mg/mL bovine serum albumin (BSA), 5 microliters of sample plasmid: Tet-pLKO-puro.
  • BSA bovine serum albumin
  • the fluid channel of the upper chip and the microwell array of the lower chip are staggered by manual relative position movement, and the liquid in the microwell array of the lower chip forms a PCR solution microdroplet microwell array.
  • the chip is placed on an inverted fluorescence microscope (Nikon Ti-2) for photographing and fluorescence measurement.
  • the fluorescence detection signal of the FAM channel is used to determine whether there is gene amplification. If the microwell contains the target gene fragment, there will be a significant increase in fluorescence after thermal cycling.
  • the experimental data analyzes the changes in the fluorescence signal of three adjacent microwells before (as shown in Figure 5) and after (as shown in Figure 6).
  • the signal peak of one of the microwells is significantly enhanced, while the other two Basically remain unchanged, indicating that one of the microwells contained the target gene fragment and was subjected to PCR amplification, while the other two microwells did not contain the target gene fragment, so there was no change after PCR amplification.
  • the method of the present invention can ensure that each microwell contains at most one target gene fragment, and cross-contamination between the microwells is not easy to occur, which provides a basis for the accuracy of quantitative detection such as digital PCR.
  • the method provided by the present invention can also design an expansion channel on the upper chip (as shown in Fig. 7).
  • the expansion channel can be the same depth, shallower or deeper as the fluid channel; the width can also be the same or different from the fluid channel.
  • the expansion channel can be filled with air or organic phase liquid.
  • the expansion channel provides space for the expansion of the aqueous solution, which further ensures the expansion of the micropores. There will be no cross-contamination during the reaction.
  • Example 3 Displacement microfluidic chip No. 2
  • the structure of the upper chip is the same as that in embodiment 1, except that: the lower chip is provided with an array of micro-holes with progressively increasing diameters, including: 4000 micro-holes are divided into four columns, each column 1000, the diameter of the micropores from left to right are: 60 micrometers, 100 micrometers, 250 micrometers, 500 micrometers, and the depth is 25 micrometers.
  • the chip preparation can be the same as in Example 1.
  • the upper chip and the lower chip are prepared on the glass material by wet etching.
  • the width of the fluid channel of the upper chip is 10 mm, the length is 25 mm, and the depth is 50 ⁇ m.
  • the upper chip contains a liquid inlet and a liquid outlet.
  • the volume of the various shaped micropores is 0.01-100 nanoliters.
  • the micropores are distributed in an area of 4.5 mm wide and 12.5 mm long.
  • the aspect ratio (depth/width) of the micropit after the microhole of the lower chip is etched is preferably less than 1, and more preferably ⁇ 1/2.
  • the structure of the upper chip is the same as that in embodiment 1, except that: the lower chip is provided with a micro-hole array composed of special-shaped micro-holes, including: round, rectangular, square, cross, and triangle. .
  • the chip preparation can be the same as in Example 1.
  • the upper chip and the lower chip are prepared on the glass material by wet etching.
  • the width of the fluid channel of the upper chip is 5 mm, the length is 15 mm, and the depth is 50 ⁇ m.
  • the upper chip contains a liquid inlet and a liquid outlet.
  • the volume of each of the micropores may be 0.1-100 nanoliters or 1-50 nanoliters.
  • the aspect ratio (depth/width) of the micropits after the microholes of the lower chip is etched is preferably less than 1.
  • the structure of the upper chip is the same as that in the first embodiment.
  • the irregular micro-holes on the lower chip include round holes, stepped round holes, and stepped square holes.
  • the diameter of the circular hole is 80 microns, and the depth is 25 microns;
  • the diameter of the first-level circular hole of the step circular hole is 10-1000 microns, and the depth is 2-200 microns, and the diameter of the second-level circular hole is 5-500 microns, and the depth is 1-100 microns;
  • the stepped square hole penetrates the lower chip, the first-level rectangle is 10-1000 microns in length, 10-1000 microns in width, and 2-200 microns in depth, and the second-level rectangle is 5-500 microns in length and 5-500 microns in width. , 1-100 microns deep.
  • the chip preparation can be the same as in Example 1.
  • the upper chip and the lower chip are prepared on the glass material by wet etching.
  • the width of the fluid channel of the upper chip is 5 mm, the length is 15 mm, and the depth is 50 ⁇ m.
  • the upper chip contains a liquid inlet and a liquid outlet.
  • the lower chip in this embodiment The volume of each shaped micropore is 0.1-100 nanoliters.
  • the micropores are distributed in an area with a width of 5 mm and a length of 15 mm.
  • the aspect ratio (depth/width) of the micropit after the microhole of the lower chip is etched is preferably less than 0.5.
  • the structure of the upper chip is the same as that in embodiment 1.
  • the lower chip is provided with an array of micropores with the same diameter. 3000 micropores are divided into 10 columns, 300 in each column, and the diameter of the micropores is 250 Micrometers, the depth is 25 micrometers.
  • the chip preparation can be the same as in Example 1.
  • the upper chip and the lower chip are prepared on the glass material by wet etching.
  • the process includes: assembling the upper chip A and the lower chip B to form a displacement microfluidic chip (C), then loading the sample (D), sliding to form a droplet array (E), and then performing the process on the microfluidic chip Incubation and detection.
  • a displacement microfluidic chip C
  • D sample
  • E droplet array
  • Fig. 18 shows the result of forming a droplet array of this embodiment.
  • Example 2 the displacement microfluidic chip No.1 prepared in Example 1 was used, and the digital PCR reaction was performed using the same method as in Example 2, and the digital PCR reaction results were compared with those obtained by Stilla's Naica TM Crystal The detection results of the droplet digital PCR system were compared.
  • the fluid channel of the upper chip and the microwell array of the lower chip are staggered by manual relative position movement, and the liquid in the microwell array of the lower chip forms a PCR solution microdroplet microwell array.
  • the chip is placed on an inverted fluorescence microscope (Nikon Ti-2) for photographing and fluorescence measurement.
  • the fluorescence detection signal of the FAM channel is used to determine whether there is gene amplification. If there is gene amplification, there is obvious fluorescence signal enhancement in the microwell, which is defined as a positive spot.
  • the number of positive spots and the number of total microfluidics in the experiment can be calculated by the principle of Poisson distribution statistics for their initial nucleic acid concentration.
  • This example and Stilla's digital PCR quantification effect achieved good consistency in 3 different concentrations (10fg/ ⁇ l, 1fg/ ⁇ l, 0.1fg/ ⁇ l) of nucleic acid.
  • Figure 19 shows the quantitative results of the digital PCR of this example.

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Abstract

本发明提供一种在微流控芯片上生成液滴阵列的方法，包括以下步骤：将上芯片和下芯片组装到初始位置，该上芯片的流体管道将该下芯片的微孔阵列部分或者全部覆盖；将溶液注入到芯片中，该溶液部分或全部充满该下芯片的微孔阵列；将该上芯片和该下芯片相对移动到液体分割位置，该上芯片的流体管道与该下芯片的微孔阵列不再重叠，该溶液分散到该微孔阵列中，形成液滴阵列。该上芯片和下芯片间接触面是疏水的，且该微孔阵列将生成的液滴之间充分物理隔离开，避免了交叉污染。本发明能对生成液滴的大小、形态进行有效控制。

Description

一种在微流控芯片上生成液滴阵列的方法和装置 技术领域
本发明涉及液滴生成领域，尤其涉及一种在微流控芯片上生成液滴阵列的方法和装置。
背景技术
液滴(droplet)在物理、化学、生物和医学方面都有非常广泛的应用。在这其中大量的(通常100个以上的)液滴阵列在基因、蛋白及细胞分析方面展现了独特的优势。
在这其中，数字PCR基因扩增就是基于大量独立微液滴的一种对目标基因进行精准定量的技术。其通过将反应溶液分散到皮升或纳升的微液滴或反应微孔中，达到每一个微液滴或微孔中至多包含一个拷贝的目标基因。例如有些微液滴或微孔包含一个目标基因，另外的微液滴或微孔不包含目标基因。通过在微液滴或微孔中对目标基因的特异性扩增，使其产生可检测的信号，例如荧光信号。通过统计有信号增强的微液滴占总液滴数的比例(或者统计没有信号增强的微液滴占总液滴数的比例)即可根据泊松分布等统计学计算方法精确的计算出目标基因的初始浓度。
目前数字PCR定量的方法主要分为流动生成微液滴法和基于反应微孔的芯片法。这两种方法都是通过将反应溶液分散到大量的微反应单元中，达到数字PCR的目的。
流动生成液滴法主要是通过特殊的微流控流体通道的设计，用有机液体将水溶液截断生成一系列的液滴。这种方法的描述请参考Angew.Chem.Int.Ed.2006,45,7336–7356。流体法的代表之一是交叉流动的液滴形成(cross-flowing droplet formation)。这种方法是通过有机相和水相以彼此成角度(T型或Y型)的流动，利用剪切力(shear force)将水相拉伸并最终生成液滴。另外一种代表的方法是流动聚焦液滴形成(flow focusing droplet formation)。这种方法通过有机项和水相的非平行流动，经过一个约束的狭窄区域而生成液滴。另外一种方法是共同流动的液滴生成(Co-flowing droplet formation)，这种方法是使分散相(例如水相)通道被封闭在连续相(例如有机相)通道内，在分散相通道的末端，流体被拉伸直至剪切力将其断裂形 成液滴。以上所描述的这些方法已经有了一些商品化的产品，其中具有代表性的是伯乐(BioRad)的液滴式数字PCR系统(ddPCR)。伯乐的液滴式数字PCR系统具备有一套液滴生成芯片，可以相对快速的生成上万个纳升级的微液滴。将微液滴在热循环仪中进行扩增，在通过一套类似于流式检测的液体荧光检测系统对微液滴的荧光进行检测。
芯片法主要是通过在微流控芯片上形成微孔或微反应池，再将分散相水溶液分散到微孔或微反应池中，使微孔中的水相形成相对独立的微液滴。这其中比较具有代表性的是赛默飞(Thermo)的QuantStudio 3D数字PCR系统。赛默飞的系统具有一片包含上万个反应微孔的微流控芯片，将反应溶液分散到这些微孔中，再用有机相(油相)将这些微孔覆盖形成独立的反应微孔。
滑动芯片(SlipChip)是一种微流控芯片，其上子芯片的下表面及下子芯片的上表面制备有大量的微孔。在初始位置，将上下子芯片装配到一起，上下子芯片的微孔有部分叠加，从而形成连通的流体通道。待溶液注射到芯片中后，将上下子芯片相对滑动，微孔不再相互部分叠加，从而产生大量的液滴。这一方法需要上子芯片的大量微孔与下子芯片的大量微孔在初始位置进行精准的对齐操作，才能保证液滴顺利的加入到微孔中去。
现有技术存在的缺点主要包括以下几点：流动生成液滴法为了生成大小均一度较好的液滴，液滴法需要对不互溶的两个液体的流速进行精准的控制，这一过程通常需要借助流体泵等仪器，整体的仪器系统会比较复杂，仪器体积交大，系统较为昂贵，而且液滴的均一性对其进行数字PCR等分析结果的准确性和可靠性至关重要，另外为了保证生成的液滴之间没有交叉污染(物质分子在液滴间的传递)以及避免液滴之间的融合(两个或多个液滴相互接触，变为一个更大的液滴)，通常需要使用表面活性剂(surfactant)。表面活性剂通常价格较高，而且会影响水溶液内的生物化学反应。QuantStudio 3D数字PCR系统则需要很多手动操作步骤，生成液滴过程较复杂，并且其是通过有机相(油相)将微孔中的水溶液进行分割，容易产生微孔之间的交叉污染。而Stephen Quake研究的微阀控制的微流控芯片也是需要复杂的压力控制系统(去进行微阀的控制)，另外芯片(耗材)的加工成本非常高；滑动芯片(SlipChip)这一方法需要上子芯片的大量微孔与下子芯片的大量微孔在初始位置进行精准的对齐操作，才能保证液滴顺利的加入到微孔中去，且对芯片加工，组装和控制的要求较高。
因此，本领域的技术人员致力于开发一种在微流控芯片上生成液滴阵列的方法，通过简单的上下芯片组合的装置和简便的操作方法即可有效可控地形成 液滴阵列，并通过物理隔离方式简便、有效、充分地避免交叉污染现象。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何提供一种在微流控芯片上生成液滴阵列的方法，通过简单的上下芯片组合的装置和简便的操作方法即可有效可控地形成液滴阵列，并通过物理隔离方式简便、有效、充分地避免交叉污染现象，克服现有技术的不足。
在本发明的第一方面，提供了一种在微流控芯片上生成液滴阵列的方法，包括以下步骤：
步骤一、将上芯片和下芯片组装到初始位置，所述上芯片的流体管道所述下芯片的微孔阵列部分或者全部覆盖；所述上芯片的流体管道为包含一个或多个连通的流体通道的结构；
步骤二、将溶液注入到芯片中，所述溶液部分或全部充满所述下芯片的微孔阵列；
步骤三、将所述上芯片和所述下芯片相对移动到液体分割位置，所述上芯片的流体管道与所述下芯片的微孔阵列不再重叠，所述溶液分散到所述微孔阵列中，形成液滴阵列。
进一步地，所述微流控芯片包括所述上芯片和所述下芯片，其中所述上芯片的下表面与所述下芯片的上表面相互接触，且相互接触的所述上芯片的下表面与所述下芯片的上表面需进行疏水化改性处理；所述上芯片或所述下芯片上设置有进液孔，且所述上芯片或所述下芯片上还可设置出液孔。
进一步地，所述上芯片的流体通道的性质可选择直线型、曲线型或二者的组合设计。
进一步地，所述上芯片的流体通道的尺寸规格范围为：宽度为1μm～10cm，长度为100μm～100cm，深度为1μm～1cm。
进一步地，所述上芯片的流体通道的表面需进行疏水化或亲水化改性处理。
进一步地，所述下芯片的微孔阵列可包含一个或多个微孔，所述微孔的大小和深度可设计一致、也可不同；所述微孔的表面需进行表面改性处理，所述表面改性处理可选自物理改性、化学改性、生物改性中的一种或几种。
进一步地，所述步骤一中将所述上芯片与所述下芯片组装到所述初始位置 后，可先将有机相注入到芯片中，所述有机相包含疏水化改性处理表面化学成分。
进一步地，所述上芯片与所述下芯片的材质可选自玻璃、石英、塑料、陶瓷、纸质材料中的任意一种。
进一步地，所述上芯片与所述下芯片可通过光刻蚀、氢氟酸湿法刻蚀、干法刻蚀、热压花法制备。
进一步地，所述上芯片上还可设计一个或多个扩展通道，所述扩展通道中可填充空气或有机相，且当所述上芯片和所述下芯片相对移动到所述液体分割位置时，所述扩展通道与所述下芯片的微孔阵列重叠。
在本发明的第二方面，提供了一种用于生成液滴阵列的微流控芯片，所述芯片包括：
上芯片，所述上芯片的流体管道为包含一个或多个连通的流体通道的结构；和
下芯片，所述的下芯片设有微孔阵列；
其中，当将上芯片和下芯片组装到初始位置时，所述上芯片的流体管道将所述下芯片的微孔阵列部分或者全部覆盖；
并且，当将溶液注入到芯片中，所述溶液部分或全部充满所述下芯片的微孔阵列；再将所述上芯片和所述下芯片相对移动到液体分割位置，所述上芯片的流体管道与所述下芯片的微孔阵列不再重叠，从而将所述溶液分散到所述微孔阵列中，形成液滴阵列。
在另一优选例中，所述上芯片的下表面与所述下芯片的上表面相互接触。
在另一优选例中，所述相互接触的所述上芯片的下表面与所述下芯片的上表面是经疏水化改性处理的。
在另一优选例中，所述上芯片或所述下芯片上设置有进液孔，且所述上芯片或所述下芯片上还可设置出液孔。
在另一优选例中，所述上芯片的流体通道的性质为直线型、曲线型或二者的组合设计。
在另一优选例中，所述上芯片的流体通道的尺寸规格范围为：宽度为1μm～10cm，长度为100μm～100cm，深度为1μm～1cm。
在另一优选例中，所述上芯片的流体通道的表面是经疏水化或亲水化改性处理的。
在另一优选例中，所述下芯片的微孔阵列可包含多个微孔。
在另一优选例中，所述微孔的表面是经表面改性处理的。
在另一优选例中，所述表面改性处理可选自物理改性、化学改性、生物改性中的一种或几种。
在另一优选例中，所述上芯片与所述下芯片的材质选自下组：玻璃、石英、塑料、陶瓷、纸质材料、或其组合。
在另一优选例中，所述上芯片与所述下芯片的材质选自下组：玻璃、石英、塑料、陶瓷、或其组合。
在另一优选例中，所述上芯片与所述下芯片是通过光刻蚀、氢氟酸湿法刻蚀、干法刻蚀、热压花法、注塑成型、3D打印制备的。
在另一优选例中，所述上芯片上设有一个或多个扩展通道，所述扩展通道中填充空气或有机相，且当所述上芯片和所述下芯片相对移动到所述液体分割位置时，所述扩展通道与所述下芯片的微孔阵列重叠。
在另一优选例中，所述的微孔阵列中，微孔密度为4-100000孔/cm 2，较佳地9-9000孔/cm 2，更佳地25-5000孔/cm 2或100-5000孔/cm 2。
在另一优选例中，所述各微孔的容积为0.001-100纳升，较佳地0.01-50纳升，更佳地0.05-10纳升，最佳地0.1-5纳升。
在另一优选例中，所述各微孔的深度D与截面积S 1/2之比(D/S 1/2)为1/200至1，较佳地1/20-0.8，更佳地1/5-0.5。
在另一优选例中，当微孔截面为正方形时，各微孔的深度D与所述正方形的长度A之比(D/A)为1/200至1，较佳地1/20-0.8，更佳地1/5-0.5。
在另一优选例中，所述的微孔的深度D为5-200微米，较佳地10-100微米，更佳地20-50微米。
在另一优选例中，当微孔截面为圆形时，各微孔的深度D与所述圆形的长度d之比(D/d)为1/200至1，较佳地1/20-0.8，更佳地1/5-0.5。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
与现有技术相比，本发明至少具有以下有益技术效果：
(1)本发明与传统的滑动芯片方法相比，并不需要在上芯片和下芯片的微 孔的部分重叠建立联通的流体通道，在芯片加工方面更为简单，可以允许更大的公差，在芯片的组装方面也不再需要精密的对齐操作，组装更为便捷；
(2)本发明相对于其他液滴生成方法不需要复杂的控制系统，且可对生成液滴的大小、形态等进行有效的控制；
(3)本发明较传统的微孔阵列微流控芯片相比可以进行很好的物理隔离，使微孔之间不存在交叉污染。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例的上下芯片移动后的位置组合图；
图2是本发明的一个较佳实施例的上下芯片初始位置组合图；
图3是本发明的一个较佳实施例的上芯片仰视图；
图4是本发明的一个较佳实施例的下芯片俯视图；
图5是本发明的一个较佳实施例的数字PCR扩增前三个相邻微孔的荧光信号图；
图6是本发明的一个较佳实施例的数字PCR扩增后三个相邻微孔的荧光信号图；
图7是本发明的一个较佳实施例的具有扩展通道的上芯片示意图；
图8是本发明的一个较佳实施例的具有扩展通道的上芯片与下芯片初始位置组合图；
图9是本发明的一个较佳实施例的具有扩展通道的上芯片与下芯片移动后的位置组合图；
图10是本发明的另一较佳实施例的下芯片俯视图，其中设有不同大小的微孔；
图11是本发明的另一较佳实施例的具有扩展通道的上芯片与下芯片初始位置组合图；
图12是本发明的另一较佳实施例的具有扩展通道的上芯片与下芯片移动后的位置组合图；
图13是本发明的另一较佳实施例的下芯片的俯视图，其中设有不同形状的微孔。
图14是具有扩展通道的上芯片与图13所示下芯片处于初始位置的组合图；
图15是具有扩展通道的上芯片与图13所示下芯片移动后的处于液体分割位置的组合图；
图16显示了本发明另一优选例的下芯片示意图，其中，在一个微单元里可以设有不同的微结构。
图17是数字PCR中具有扩展通道的上芯片与下芯片移动后的反应过程；
图18显示了本发明另一优选例的生成大小均一的微孔阵列。
图19显示了采用基于本发明微流控芯片的数字PCR系统所获得的核酸定量检测结果。
具体实施方式
本发明人经过广泛而深入的研究，经过大量筛选，首次开发了一种结构独特的位移式微流控芯片。本发明位移式微流控芯片，通过上芯片和下芯片的相对滑动，即从初始位置滑动到液体分割位置时，可快速、高效、简便地将注入芯片的溶液(如数字PCR的反应液)分散到下芯片的微孔阵列中，形成液滴阵列。在此基础上完成了本发明。
术语
如本文所用，术语“上芯片”和“上芯片板”可互换使用。
如本文所用，术语“下芯片”和“下芯片板”可互换使用。
应理解，为了便于描述，“上”、“下”、“左”、“右”是相对的，他们被用于表述相对的空间位置关系。例如，上芯片也可被称为下芯片，而下芯片也可被称为上芯片。
如本文所用，术语“在…之间”，当用于在“第一位置”和“第二位置”之间运动的上下文中时，可以指仅仅从第一位置向第二位置运动，仅仅从第二位置向第一位置运动，或者从第一位置向第二位置和从第二位置向第一位置运动。典型地，第一位置为上芯片和下芯片进行组装的初始位置，或向芯片注入流体(如溶液)时的上芯片和下芯片所处的注入位置；第二位置为流体分割位置。
微流控芯片
如本文所用，术语“本发明的芯片”、“本发明的微流控芯片”、“位移式微流控芯片”、“本发明的位移式微流控芯片”可互换使用，指本发明的第 二方面中所述的微流控芯片，所述芯片通过上芯片和下芯片的相对滑动，即从初始位置滑动到液体分割位置时，可将注入芯片的溶液分散到下芯片的微孔阵列中，形成液滴阵列。
本发明位移式微流控芯片包括互相配合使用的“上芯片”和“下芯片”。
在本发明中，所述的上芯片包括一个或多个连通的流体通道的结构，流体通道的尺寸规格范围为：宽度为1μm～10cm，长度为100μm～100cm，深度为1μm～1cm。
本发明的微流控芯片可以用来生成不同大小和形状的液滴阵列。
在另一优选例中，所述上芯片设有进液孔。
在另一优选例中，所述上芯片还可设置出液孔。
在另一优选例中，所述上芯片上设有一个或多个扩展通道，所述扩展通道中填充空气或有机相。芯片的上层扩展通道中的溶液可以作为额外的反应溶液，提高总体的反应液体积，从而达到提高反应灵敏度的目的。在本发明中，所述下芯片设有微孔阵列。在本发明中，微孔密度没有特别限制。典型地，微孔密度为4-100,000孔/cm 2，较佳地9-9000孔/cm 2，更佳地25-5000孔/cm 2或100-5000孔/cm 2。
所述微孔的大小和深度可设计一致、也可不同。
在本发明中，下芯片可以包含不同大小的微孔(well)，用来生成容积不同的液体单元。
在另一优选例中，下芯片也可以包含深度不同的微孔，生成不同深度的液体单元。
在另一优选例中，下芯片也可以包含不同形状的微坑，代表性的形状包括(但并不限于)：圆形、矩形、正方形、十字形、三角形、或其他任意形状。
在本发明中，所述的所述微孔的表面可以是未经表面处理的，也可以是经表面改性处理的。代表性的表面改性处理包括(但并不限于)：物理改性、化学改性、生物改性、或其组合。
优选地，本发明芯片的表面使用气相硅烷化的方法进行改性，例如，使用二甲基二氯硅烷，对玻璃表面进行疏水化改性处理。
生成液滴阵列的方法
本发明还提供了基于本发明的位移式微流控芯片，生成液滴阵列的方法。
典型地，该方法包括：
步骤一、提供本发明的位移式微流控芯片，其中，上芯片和下芯片处于初始位置；此时，所述上芯片的流体管道将所述下芯片的微孔阵列部分或者全部覆盖；
步骤二、将溶液注入到芯片中，从而使得所述溶液部分或全部充满所述下芯片的微孔阵列；
步骤三、将所述上芯片和所述下芯片相对移动(或滑动)到液体分割位置，此时，所述上芯片的流体管道与所述下芯片的微孔阵列不再重叠，从而使得所述溶液分散到所述微孔阵列中，形成液滴阵列。
优选地，当本发明的位移式微流控芯片设有扩展通道，且扩展通道含有有机相成分时，代表性的有机相为矿物油和十四烷的混合物，例如以等体积混合形成的混合物。当上下芯片中间加有一层有机相液体，组合在加样初始位置时，上芯片的连通的流体通道和下芯片的微孔被有机相所占据。
应用
本发明还提供了本发明位移式微流控芯片以及生成液滴阵列的应用。
采用本发明的微流控芯片，通过对上芯片和下芯片的简便操作，可有效可控地形成包含大量微液滴的阵列(如1000-10000或更多个微液滴)。
本发明的装置和方法可应用于需要大量独立的微液体的应用场合，一种典型的应用是将本发明的位移式微流控芯片用于数字PCR反应，从而对核酸样品进行定量检测。
本发明的主要优点包括：
(a)通过简单的上下芯片组合的装置和简便的操作方法即可有效可控地形成微液滴阵列，并通过物理隔离方式简便、有效、充分地避免交叉污染现象。
(b)本发明不需要在上芯片和下芯片的微孔的部分重叠建立联通的流体通道，在芯片加工方面更为简单，可以允许更大的公差，在芯片的组装方面也不再需要精密的对齐操作，组装更为便捷。
(c)本发明相对于其他液滴生成方法不需要复杂的控制系统，且可对生成液滴的大小、形态等进行有效的控制。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方 法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1、位移式微流控芯片No.1的制备和生成大小均一的微液滴阵列
本实施例通过湿法刻蚀的方法在玻璃材料上制备上芯片(如图3所示)和下芯片(如图4所示)。上芯片的流体通道的宽度为5毫米，长度为15毫米，深度为50微米。上芯片包含一个液体入口，一个液体出口。
为了更方便地显示结构特征，该微流控芯片的结构示意图如图3和图4所示。然而，实际制备的下芯片包含5000个微孔，分布在4.5毫米宽12.5长的区域内。下芯片的微孔刻蚀后直径为80微米，深度为25微米。
芯片的表面使用气相硅烷化的方法，使用二甲基二氯硅烷，对玻璃表面进行疏水化改性处理。有机相成分为矿物油和十四烷以等体积进行混合。
将所述上芯片与所述下芯片组装后，所述上下芯片中间加有一层有机相液体，并将二者的相对位置摆放在如图2所示的加样初始位置，上芯片的连通的流体通道和下芯片的微孔被有机相所占据。通过上芯片的进样口，将含有聚乙二醇辛基苯基醚和荧光素的水溶液打入芯片中。水溶液会将流体通道和微孔中的有机相置换掉。
通过手动的相对位置移动，将上芯片的流体通道与下芯片的微孔阵列错开到如图1所示的液体分割位置，在下芯片的微孔阵列中的液体形成了微液滴微孔阵列。
通过荧光显微镜(Nikon Ti-2)对微孔中的液滴进行荧光拍照并通过Nikon的分析软件对液滴的大小进行分析。所生成液滴的直径均值为74微米，并且其标准差小于5％(约100个液滴测量值)。这证明了本发明提出的方法可以用来生成大小均一的液滴阵列。
实施例2、数字PCR实验
芯片制备与实施例1相同。通过湿法刻蚀的方法在玻璃材料上制备上芯片和下芯片。上芯片的流体通道的宽度为5毫米，长度为15毫米，深度为50微米。上芯片包含一个液体入口，一个液体出口。下芯片包含5000个微孔分布在4.5毫米宽12.5长的区域内。下芯片的微孔刻蚀后直径为80微米，深度为25微米。芯片的表面使用气相硅烷化的方法，使用二甲基二氯硅烷，对玻璃表面进行疏水化改性处理。有机相成分为矿物油和十四烷以等体积进行混合。上下芯片中 间加有一层有机相液体，组合在加样初始位置时，上芯片的连通的流体通道和下芯片的微孔被有机相所占据。
PCR反应溶液制备：50微升的反应试剂包括：1.25微升的引物(primer)-1：CAGCGAGTCAGTGAGCGAGGAA(SEQ ID No:1)；1.25微升的引物-2：TGTAAAGCCTGGGGTGCCTAA(SEQ ID No:2)；25微升的EvaGreen 2x PCR反应溶液(购买自伯乐公司)；15微升PCR试剂用水；2.5微升10mg/mL牛血清白蛋白(BSA)，5微升样品plasmid:Tet-pLKO-puro。
PCR反应溶液打入芯片后，通过手动的相对位置移动，将上芯片的流体通道与下芯片的微孔阵列错开，在下芯片的微孔阵列中的液体形成了PCR溶液微液滴微孔阵列。将芯片放置于平板PCR仪上，设置扩增温度为1分钟在95℃，30秒在55℃，30秒在72℃，并反复循环40次。
热循环完成后，芯片被放置在倒置式荧光显微镜(Nikon Ti-2)上进行拍照和荧光测量。FAM通道的荧光检测信号被用来判断是否有基因扩增。如果微孔中包含目标基因片段，热循环后会有显著的荧光增强。实验数据对三个相邻微孔的荧光信号测量前(如图5所示)后(如图6所示)变化情况进行分析，可发现其中一个微孔的信号峰值明显增强，而另外两个基本保持不变，说明其中一个微孔中包含目标基因片段，并进行了PCR扩增，而另外两个微孔中不含目标基因片段，故PCR扩增后无变化。这说明了本发明的方法可保证每个微孔中至多含有一个目标基因片段，且微孔之间不易发生交叉污染，为数字PCR等定量检测的精准性提供了依据。
本发明提供的方法还可在上芯片上设计扩展通道(如图7所示)。所述扩展通道可以和流体通道深度相同，更浅或者更深；宽度也可以与流体通道相同或不同。所述扩展通道中可填充空气或有机相液体。将具有扩展通道的上芯片与下芯片组装成如图8所示的初始位置后，注入水溶液，部分或全部充满下芯片的微孔。之后，再将上芯片与下芯片相对移动到如图9所示的液体分割位置，液滴阵列形成，且液滴间被物理隔离，同时所述扩展通道与所述下芯片的微孔阵列重叠，为微孔中的水溶液提供了额外的扩展空间，在一些有温度变化的过程中，例如温度的上升，水溶液会膨胀，所述扩展通道为水溶液的膨胀提供了空间，进一步保证了微孔之间在反应过程中不会有交叉污染。
实施例3、位移式微流控芯片No.2
参见图10、11和12。在本实施例中，上芯片的结构与实施例1中的相同， 不同之处在于：下芯片上设有直径逐级递增的微孔阵列，包括：4000个微孔分为四列，每列1000个，微孔直径从左至右依次为：60微米、100微米、250微米、500微米，深度为25微米。
该芯片制备可与实施例1相同。通过湿法刻蚀的方法在玻璃材料上制备上芯片和下芯片。
在本实施例中，上芯片的流体通道的宽度为10毫米，长度为25毫米，深度为50微米。上芯片包含一个液体入口，一个液体出口。
在本实施例中的下芯片中，各异形微孔的容积为0.01-100纳升。所述微孔分布在4.5毫米宽12.5毫米长的区域内。
下芯片的微孔刻蚀后微坑的深宽比(深度/宽度)优选地小于1，更佳地≤1/2。
实施例4、位移式微流控芯片No.3
参见图13、14和15。在本实施例中，上芯片的结构与实施例1中的相同，不同之处在于：下芯片上设有异形微孔构成的微孔阵列，包括：圆形、矩形、正方形、十字形、三角形。
该芯片制备可与实施例1相同。通过湿法刻蚀的方法在玻璃材料上制备上芯片和下芯片。
在本实施例中，上芯片的流体通道的宽度为5毫米，长度为15毫米，深度为50微米。上芯片包含一个液体入口，一个液体出口。
在本实施例中，所述各微孔的容积可以为0.1-100纳升或1-50纳升。
下芯片的微孔刻蚀后微坑的深宽比(深度/宽度)优选地在小于1。
实施例5、位移式微流控芯片No.4
参见图16。在本实施例中，上芯片的结构与实施例1中的相同，不同之处在于：下芯片上异形微孔，包括：圆孔、阶梯圆孔、阶梯方孔。刻蚀后圆孔直径为80微米，深度为25微米；阶梯圆孔的第一级圆孔直径为10-1000微米，深2-200微米，第二级圆孔直径为5-500微米，深1-100微米；阶梯方孔部分贯穿下芯片，第一级矩形长10-1000微米，宽10-1000微米，深2-200微米，第二级矩形长5-500微米，宽5-500微米，深1-100微米。
该芯片制备可与实施例1相同。通过湿法刻蚀的方法在玻璃材料上制备上芯片和下芯片。
在本实施例中，上芯片的流体通道的宽度为5毫米，长度为15毫米，深度为50微米。上芯片包含一个液体入口，一个液体出口。
本实施例中的下芯片。各异形微孔的容积为0.1-100纳升。所述微孔分布在5毫米宽15长的区域内。
下芯片的微孔刻蚀后微坑的深宽比(深度/宽度)优选地小于0.5。
实施例6、位移式微流控芯片No.5
在本实施例中，上芯片的结构与实施例1中的相同，下芯片上设有直径相同的微孔阵列，3000个微孔分为10列，每列300个，微孔直径为：250微米，深度为25微米。
该芯片制备可与实施例1相同。通过湿法刻蚀的方法在玻璃材料上制备上芯片和下芯片。
用该微流控芯片No.5，采用图17所示的流程生成液滴阵列并进行数字PCR检测：
其中所述流程包括：将上芯片A和下芯片B进行组装，形成位移式微流控芯片(C)，然后上样(D)，滑动形成液滴阵列(E)，然后对微流控芯片进行孵育和检测。其中，在图C、D和E下方给出了相应各自状态下的微流控芯片的剖视图。
图18显示了该实施例的形成液滴阵列的结果。
实施例7数字PCR应用
在本实施例中采用实施例1中制备的位移式微流控芯片No.1，并采用与实施例2相同的方法进行数字PCR反应，并将数字PCR的反应结果与通过Stilla公司的Naica TM Crystal微滴数字PCR系统的检测结果进行了对比。
PCR反应溶液打入芯片后，通过手动的相对位置移动，将上芯片的流体通道与下芯片的微孔阵列错开，在下芯片的微孔阵列中的液体形成了PCR溶液微液滴微孔阵列。将芯片放置于平板PCR仪上，设置扩增温度为1分钟在95℃，30秒在55℃，30秒在72℃，并反复循环40次。
热循环完成后，芯片被放置在倒置式荧光显微镜(Nikon Ti-2)上进行拍照和荧光测量。FAM通道的荧光检测信号被用来判断是否有基因扩增。有基因扩增则微孔中有明显的荧光信号增强，则定义为阳性点。
实验中阳性点的数量和总微流控的数量可以通过泊松分布统计学原理对其初始的核酸浓度进行计算。本实施例与Stilla公司的数字PCR定量效果在3个 不同浓度(10fg/μl,1fg/μl,0.1fg/μl)的核酸都取得了很好的一致性。
图19显示了该实施例的数字PCR的定量结果。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (20)

  1. 一种在微流控芯片上生成液滴阵列的方法，其特征在于，包括以下步骤：
    步骤一、将上芯片和下芯片组装到初始位置，所述上芯片的流体管道将所述下芯片的微孔阵列部分或者全部覆盖；所述上芯片的流体管道为包含一个或多个连通的流体通道的结构；
    步骤二、将溶液注入到芯片中，所述溶液部分或全部充满所述下芯片的微孔阵列；
    步骤三、将所述上芯片和所述下芯片相对移动到液体分割位置，所述上芯片的流体管道与所述下芯片的微孔阵列不再重叠，所述溶液分散到所述微孔阵列中，形成液滴阵列。
  2. 如权利要求1所述的方法，其特征在于，所述微流控芯片包括所述上芯片和所述下芯片，其中所述上芯片的下表面与所述下芯片的上表面相互接触，且相互接触的所述上芯片的下表面与所述下芯片的上表面需进行疏水化改性处理；所述上芯片或所述下芯片上设置有进液孔，且所述上芯片或所述下芯片上还可设置出液孔。
  3. 如权利要求1所述的方法，其特征在于，所述上芯片的流体通道的性质可选择直线型、曲线型或二者的组合设计。
  4. 如权利要求1所述的方法，其特征在于，所述上芯片的流体通道的尺寸规格范围为：宽度为1μm～10cm，长度为100μm～100cm，深度为1μm～1cm。
  5. 如权利要求1所述的方法，其特征在于，所述上芯片的流体通道的表面需进行疏水化或亲水化改性处理。
  6. 如权利要求1所述的方法，其特征在于，所述下芯片的微孔阵列可包含一个或多个微孔，所述微孔的大小和深度可设计一致、也可不同；所述微孔的表面需进行表面改性处理，所述表面改性处理可选自物理改性、化学改性、生物改性中的一种或几种。
  7. 如权利要求1所述的方法，其特征在于，所述步骤一中将所述上芯片与所述下芯片组装到所述初始位置后，可先将有机相注入到芯片中，所述有机相包含疏水化改性处理表面化学成分。
  8. 如权利要求1所述的方法，其特征在于，所述上芯片与所述下芯片的材质可选自玻璃、石英、塑料、陶瓷、纸质材料中的任意一种。
  9. 如权利要求1所述的方法，其特征在于，所述上芯片与所述下芯片可通过光刻蚀、氢氟酸湿法刻蚀、干法刻蚀、热压花法制备。
  10. 如权利要求1所述的方法，其特征在于，所述上芯片上还可设计一个或多个 扩展通道，所述扩展通道中可填充空气或有机相，且当所述上芯片和所述下芯片相对移动到所述液体分割位置时，所述扩展通道与所述下芯片的微孔阵列重叠。
  11. 一种用于生成液滴阵列的微流控芯片，其特征在于，所述芯片包括：
    上芯片，所述上芯片的流体管道为包含一个或多个连通的流体通道的结构；和下芯片，所述的下芯片设有微孔阵列；
    其中，当将上芯片和下芯片组装到初始位置时，所述上芯片的流体管道将所述下芯片的微孔阵列部分或者全部覆盖；
    并且，当将溶液注入到芯片中，所述溶液部分或全部充满所述下芯片的微孔阵列；再将所述上芯片和所述下芯片相对移动到液体分割位置，所述上芯片的流体管道与所述下芯片的微孔阵列不再重叠，从而将所述溶液分散到所述微孔阵列中，形成液滴阵列。
  12. 如权利要求11所述的微流控芯片，其特征在于，所述上芯片的下表面与所述下芯片的上表面相互接触。
  13. 如权利要求12所述的微流控芯片，其特征在于，所述相互接触的所述上芯片的下表面与所述下芯片的上表面是经疏水化改性处理的。
  14. 如权利要求11所述的微流控芯片，其特征在于，所述上芯片的流体通道的尺寸规格范围为：宽度为1μm～10cm，长度为100μm～100cm，深度为1μm～1cm。
  15. 如权利要求11所述的微流控芯片，其特征在于，所述上芯片的流体通道的表面是经疏水化或亲水化改性处理的。
  16. 如权利要求11所述的微流控芯片，其特征在于，所述上芯片与所述下芯片的材质选自下组：玻璃、石英、塑料、陶瓷、纸质材料、或其组合。
  17. 如权利要求11所述的微流控芯片，其特征在于，所述上芯片上设有一个或多个扩展通道，所述扩展通道中填充空气或有机相，且当所述上芯片和所述下芯片相对移动到所述液体分割位置时，所述扩展通道与所述下芯片的微孔阵列重叠。
  18. 如权利要求11所述的微流控芯片，其特征在于，所述的微孔阵列中，微孔密度为4-100000孔/cm 2，较佳地9-9000孔/cm 2，更佳地25-5000孔/cm 2或100-5000孔/cm 2。
  19. 如权利要求11所述的微流控芯片，其特征在于，所述各微孔的容积为0.001-100纳升。
  20. 如权利要求11所述的微流控芯片，其特征在于，所述各微孔的深度D与截面积S 1/2之比(D/S 1/2)为1/200至1，较佳地1/20-0.8，更佳地1/5-0.5。
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