CN113083386B - Simple and rapid discretization chip for liquid sample and using method thereof - Google Patents
Simple and rapid discretization chip for liquid sample and using method thereof Download PDFInfo
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- 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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F33/00—Other mixers; Mixing plants; Combinations of mixers
- B01F33/30—Micromixers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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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
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
- G01N2001/386—Other diluting or mixing processes
Abstract
The invention discloses a simple, convenient and fast discretization chip for liquid samples and a using method thereof. When the chip is applied to the discretization operation of a liquid phase sample, the chip mainly comprises the following steps: assembling the chip, degassing, filling a sample, and stripping the cover sheet layer and the hydrophobic microstructure layer; based on the wettability difference of oil phase and water phase liquid on the surface of a structural layer and the action of liquid surface tension, the water phase liquid in the micro-pipeline is automatically broken and pushed into the connected micro-cavities by the surface tension, so that liquid samples are dispersed into the micro-cavities, and the discretization of the micro-cavities is realized. The chip can realize simple, convenient, rapid, uniform and low-cost discretization treatment of a liquid sample, and can be applied to the fields of digital biological analysis, micro-nano particle preparation and the like.
Description
Technical Field
The invention relates to the technical field of microfluidic chip analysis, in particular to a simple, convenient and rapid liquid sample discretization chip and a using method thereof.
Background
The sample discretization is an important step in biological and chemical analysis or micro-nano material synthesis and drug particle preparation, and the traditional sample discretization method comprises the following steps: spraying, ultrasonic emulsification and stirring emulsification. Although these conventional methods have the advantages of preparing discretization samples on a large scale, uniformity and repeatability of sample discretization cannot be realized, and the methods are often difficult to be applied to discretization of micro-samples, and the discretization methods based on mechanical action may damage biological samples in liquid samples, so that the methods have great limitations in practical application. In recent years, with the development of microfluidic technology, a droplet microfluidic chip becomes a mainstream platform for the current liquid sample discretization operation, and the platform mainly utilizes the instability of an interface of two immiscible fluids, so that a liquid phase containing a sample is dispersed in another immiscible liquid phase (such as an oil phase) to form a large number of droplets through the combined action of surface tension and shearing force, thereby realizing the discretization of the sample. This method requires precise, expensive fluid-driven devices to achieve precise control of the flow rates of the two liquid phases, and also requires the addition of surfactants in either the oil or water phase to prevent coalescence of adjacent droplets, which limits the application of the method to some extent. In addition to the droplet microfluidic technology, in recent years, some array microfluidic chip discretization methods are also developed internationally, without adding a surfactant, to maintain the stability of a discretized liquid sample unit, and the influence of the surfactant on an analysis result or synthesis purity is avoided, but the methods also have respective obvious defects, for example, a micro-valve array chip needs an external air pressure device and a complicated macro-micro interface, a sliding chip needs complicated manual operation and an operation skill of an operator, the effect of discretizing a liquid sample by an hydrophilic-hydrophobic pattern chip is greatly influenced by the wettability of the liquid sample to be discretized and the surface of the chip and the flow rate of a fluid, the discretization effect is unstable, large differences exist in the decomposition volumes of different liquid samples, and the oil phase is often required to be used for removing the liquid sample in a sample filling pipeline in the liquid sample discretization process of the array chip, so as to realize the independence of the liquid samples in various micro-cavities, but the micro-pipeline has small cross-section size and large oil-phase viscosity, which results in the micro-cavity flow resistance and the discretization process is long. In a word, the existing microfluidic liquid sample large-scale discretization method has great limitations in the aspects of decomposition number, simplicity and convenience in operation, discretization efficiency, application cost, reliability and the like. Therefore, a liquid sample large-scale discretization tool and a liquid sample large-scale discretization method which are simple, convenient, rapid, high in flexibility and low in cost are urgently needed to be developed, and the requirements of the fields of digital analysis, monodisperse micro-nano particle synthesis and the like on high-throughput liquid sample discretization are met.
Disclosure of Invention
In order to solve the above disadvantages of the prior art, the present invention aims to provide a simple and fast discretization chip for a liquid sample and a using method thereof, so as to solve the problems of complex operation, low discretization efficiency, high application cost and poor reliability of the existing microfluidic liquid sample large-scale discretization method.
The technical scheme for solving the technical problems is as follows: the utility model provides a simple and convenient, quick discretization chip of liquid appearance, including cover slice layer and hydrophobic micro-structure layer, the cover slice layer includes at least one inlet port and an oil storage chamber, and hydrophobic micro-structure layer includes that at least one microcavity array and the little pipeline array and at least one kind trunk line of each microcavity in every microcavity array intercommunication microcavity array.
On the basis of the technical scheme, the invention can be further improved as follows:
further, the cover sheet layer is a PDMS (polydimethylsiloxane) plate or a polyurethane plate, preferably a polydimethylsiloxane plate.
Furthermore, the microcavity array comprises a plurality of cylindrical microcavities, the number of the cylindrical microcavities can be determined according to actual requirements and the microcavities are manufactured according to a manufacturing process, the cross sections of the cylindrical microcavities are circular or regular polygons, and the geometric shapes and the sizes of all the cylindrical microcavities in each microcavity array are consistent.
Further, the arrangement mode of the micro-pipes is not unique, for example, the micro-pipes are arranged in a staggered manner or aligned manner in each row, as long as all micro-cavities in each micro-cavity array are ensured to be communicated; the cross section of the micro-pipe is smaller than the longitudinal cross section of the micro-cavity.
Furthermore, the main sampling pipeline is positioned at one end of the microcavity array or positioned around the microcavity array, and the cross section of the main sampling pipeline is larger than that of the connecting microchannel.
Furthermore, the hydrophobic microstructure layer is made of silicon, glass, polymethyl methacrylate, polydimethylsiloxane, polycarbonate, polyethylene terephthalate, cyclic olefin copolymer, polystyrene or epoxy resin, or a composite structure material taking polydimethylsiloxane or cyclic olefin copolymer as a structure surface and glass or silicon as a supporting substrate.
Further, the thickness of the cover sheet layer is more than 1 mm, preferably 1 to 5 mm.
Further, the oil storage chamber position is not specifically limited, as long as guarantee the oil storage chamber with the introduction port not communicate can, if be close to cover plate layer end, correspond the setting with the introduction port.
Further, after the cover plate layer is attached to the hydrophobic microstructure layer, the oil storage cavity is not communicated with the microcavity array and the connecting micro-pipeline.
The application method of the simple and rapid liquid sample discretization chip comprises the following steps:
(1) Preparing: aligning, fitting and assembling the cover plate layer and the hydrophobic microstructure layer, and placing the assembled chip in a vacuum container for degassing treatment;
(2) Filling a sample: taking out the chip subjected to degassing treatment in the step (1), and dropwise adding the aqueous phase liquid sample to be discretized at a sample inlet of the chip; meanwhile, oil phase liquid is dripped into the oil storage cavity;
(3) Sample discretization: and (3) after the chip sample filling in the step (2) is finished, peeling the cover plate layer of the chip from the hydrophobic microstructure layer, and realizing the discretization of the water-phase sample liquid.
Further, the degassing treatment in the step (1) is carried out for at least 30 minutes.
In conclusion, the invention has the following beneficial effects:
1. the invention provides a simple, convenient and fast discretization chip of liquid sample and its operation method, assemble the chip and carry on the vacuum degassing treatment after finishing at first, then drip aqueous phase liquid sample and oil phase liquid to be discretized at the chip sample inlet and oil storage chamber at the same time respectively, utilize the infiltration characteristic of the oil phase liquid to the surface of hydrophobic microstructure layer to combine the capillary action of the contact gap of "cover slice layer-hydrophobic microstructure layer" in the course of peeling off, drive the oil phase to pave the surface of hydrophobic microstructure layer, because the oil phase and aqueous phase liquid are to the infiltration difference of the hydrophobic surface of hydrophobic microstructure layer and aqueous phase liquid surface tension effect, the aqueous phase liquid breaks automatically in the microtube, and is pushed into the conjunctive microcavity by surface tension, cause the liquid sample to disperse into each microcavity, realize the discretization of the aqueous phase sample liquid;
2. the chip for simply, conveniently and quickly discretizing the liquid sample does not need to depend on precise micropump driving and complex surface hydrophilic and hydrophobic graphical manufacturing, does not need a complex macro-micro interface, avoids dependence of liquid sample discretization operation on professional technicians, and can simply, conveniently and quickly realize uniform and low-cost discretization treatment of the liquid sample; meanwhile, the sample discretization method based on the chip can realize almost complete discretization of a water phase liquid sample filled in the chip, and greatly reduces the loss rate of the sample compared with the conventional liquid discretization system; in addition, the method realizes spontaneous discretization based on the micro-cavity array geometric structure and the liquid surface tension effect, so that the problem that the discretization effect of the traditional liquid drop emulsifying method is easily influenced by the fluctuation of a fluid driving system is solved, the stability of discretization liquid drops is maintained without adding a surfactant, and the influence of an additional reagent on a reaction system is avoided. In a word, the liquid sample simple and rapid discretization chip provided by the invention has the advantages of simple and convenient operation, high discretization efficiency, low cost, high sample utilization rate, good monodispersity and high reliability, and can be applied to the fields of digital biological analysis, micro-nano particle preparation synthesis and the like.
Drawings
FIG. 1 is a schematic diagram of a simple and fast discretization chip structure for liquid samples according to the present invention;
FIG. 2 is a schematic diagram showing a flow of the simple and rapid discretization chip for liquid sample application according to the present invention;
FIG. 3 is a diagram showing the experimental results of the simple and rapid discretization chip for digital PCR analysis in example 1 of the present invention;
FIG. 4 is a diagram showing the results of a digital colony counting experiment using the simple and rapid discretization chip for liquid samples in example 2 of the present invention;
FIG. 5 is a diagram showing experimental results of applying a simple and rapid discretization chip for micro-nano particle synthesis in example 3 of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1:
a simple, convenient and fast discretization chip for liquid samples is applied to digital PCR analysis, and takes EGFR L858R mutation detection in peripheral blood of a lung cancer patient as an example, the specific steps are as follows:
(1) Chip preparation: firstly, a chip die is manufactured through a photoetching process, and the specific flow is as follows: spin-coating SU8-3010 negative photoresist (500 rpm,12 rpm, 1000 rpm,40 s) on a clean 4-inch silicon wafer, standing for 1-2 h, pre-baking (95 ℃, 15 min), exposing (3 min), baking (93.5 ℃,3 min) in a dark place, naturally cooling, developing (2 min), and hard-baking (170 ℃,30 min) to obtain a first layer of a pattern structure (corresponding to a chip pipeline layer) of a chip mold; after the silicon chip is cooled, SU8-3050 negative photoresist (500 rpm,12 s, 1000 rpm,40 s) is spin-coated on the graph structure, and the graph structure is kept stand for 2-3 h. And (3) carrying out prebaking (95 ℃,30 min), exposing (3 min, 40 s), shading and postbaking (93.5 ℃,5 min), naturally cooling and developing (PGEMA, 8 min), and hard baking (170 ℃,30 min) to finish the manufacture of the graphic structure of the second layer of the chip die (corresponding to the chip micro-cavity layer), and cooling the silicon wafer to finish the manufacture of the chip die. Then, a chip hydrophobic microstructure layer is manufactured by PDMS pouring reverse mold, and the specific flow is as follows: mixing a PDMS prepolymer and a curing agent according to a mass ratio of 10:1, stirring uniformly, placing in a vacuum container, vacuumizing and defoaming for 1 h, taking out the defoamed PDMS, pouring on a chip mold, standing for 15 min, moving to a hot plate, heating and curing for 2 h at 80 ℃, cooling, peeling the cured PDMS from the mold, and cutting by using a scalpel to remove redundant PDMS to obtain a hydrophobic micro-structure layer of the chip, wherein the hydrophobic micro-structure layer comprises about 35000 micro-cavities and about 100 series micro-pipes (shown in figure 3 a), each micro-cavity is in the shape of a square column, the side length of the square of the cross section is 100 micrometers, the depth is 100 micrometers, and the width and the height of each micro-pipe are both 20 micrometers; based on the same process flow, a flat silicon wafer or a glass slide is combined with a limiting frame with the thickness of 2 mm to prepare a PDMS flat plate with the thickness of 2 mm, and a puncher and a dissection knife are used for respectively punching and cutting a rectangular through hole at a proper position to prepare a sample inlet and an oil storage cavity so as to prepare a PDMS cover plate layer; placing the prepared hydrophobic microstructure layer of the chip and a clean glass slide in a plasma cleaning machine, carrying out plasma treatment for 30 s, and bonding the non-structural surface of the hydrophobic microstructure layer with the glass slide to prepare a PDMS-glass composite hydrophobic microstructure layer; and finally, aligning and attaching the cover plate layer and the hydrophobic microstructure layer, and assembling to obtain the liquid-phase rapid discretization chip.
(2) Preparation of a sample solution: preparing a pair of specific primers aiming at EGFR L858R mutation synthesis; extracting circulating DNA from peripheral blood serum of a patient with lung cancer, adding polymerase, a primer, a probe, a buffer solution and the like into a sample tube for storing the circulating DNA, and mixing to prepare a sample solution;
(3) Degassing the chip: placing the chip obtained in the step (1) in a vacuum container for degassing treatment for at least 30 minutes, and carrying out vacuum packaging for later use;
(4) Sample introduction: dripping 40 microliters of the sample solution prepared in the step (2) into the sample inlet of the chip treated in the step (3) to form a closed micro-pipeline/micro-cavity system in the chip, absorbing air in the micro-pipeline/micro-cavity system by using a degassing PDMS cover plate to form negative pressure, and driving the aqueous phase liquid sample to enter and fill the micro-pipeline/micro-cavity system in the chip; simultaneously, silicone oil is dripped into the oil storage cavity of the cover plate layer to fill the oil storage cavity;
(5) Sample discretization: after the whole micro-pipeline/micro-cavity system in the chip is filled with the water phase liquid sample, peeling off the PDMS cover plate layer from one end close to the oil storage cavity, and driving the oil phase to be paved on the surface of the hydrophobic micro-structure layer due to the wettability of the oil phase liquid to the surface of the PDMS hydrophobic micro-structure layer and the capillary action of a contact gap between the cover plate layer and the structure layer which moves in the peeling process; meanwhile, based on the wettability difference of oil phase and water phase liquid on the hydrophobic surface of the hydrophobic microstructure layer and the surface tension effect of the water phase liquid, the water phase liquid in the micro-pipeline is automatically broken and pushed into the connected micro-cavities by the surface tension, so that a liquid sample is dispersed into each micro-cavity, and the discretization of the water phase sample liquid is realized;
(6) Sealing a liquid sample: after the discretization of the aqueous phase sample liquid is finished, covering a piece of glass sealing cover of the spin-coating PDMS prepolymer on the chip hydrophobic microstructure layer with the cover layer peeled off, so as to realize the sealing of the discretized liquid phase sample, and the formed glass-PDMS-glass sandwich structure ensures the low moisture volatilization in the PCR thermal cycle process;
(7) And (3) PCR reaction: and (4) placing the chip sealed in the step (5) on an in-situ PCR instrument for thermal cycle amplification reaction, wherein the specific thermal cycle control program is as follows: 50 ℃ for 10 minutes, 95 ℃ for 10 minutes, and 45 cycles of 95 ℃ for 45 seconds and 60 ℃ for 40 seconds;
(8) Signal acquisition and analysis: the chip after the PCR reaction in step (7) was subjected to fluorescence signal reading and data analysis by fluorescence microscopy, and the results are shown in FIG. 3b, in which (i) - (iii) of FIG. 3b show that the concentrations of the target nucleic acid molecules in the liquid phase sample are 10 respectively 4 、10 3 、10 2 PCR amplification results for copies/. Mu.L.
Example 2:
a simple, convenient and fast discretization chip of liquid sample is applied to fast counting of microbial colonies, taking escherichia coli detection as an example, and comprises the following specific steps:
(1) Chip preparation: manufacturing a chip hydrophobic microstructure layer by combining a photoetching mold manufacturing process with a PDMS (polydimethylsiloxane) pouring reverse mold and an oxygen plasma surface treatment bonding process, wherein the hydrophobic microstructure layer comprises about 40000 micro cavities and about 121000 micro pipelines for connecting adjacent micro cavities, each micro cavity is cylindrical, the diameter of each micro cavity is 100 micrometers, the depth of each micro cavity is 100 micrometers, and the width and the height of each micro pipeline are both 20 micrometers; simultaneously, a 2 mm-thick chip PDMS cover plate layer comprising a sample inlet and an oil storage cavity is manufactured through PDMS pouring and punching; then, aligning and attaching the cover sheet layer and the hydrophobic microstructure layer, and assembling to obtain a liquid-phase rapid discretization chip; specific procedures for chip preparation reference example 1;
(2) Degassing the chip: placing the chip obtained in the step (1) in a vacuum container for degassing treatment for at least 30 minutes, and carrying out vacuum packaging for later use;
(3) Sample introduction: dripping 40 microliters of the sample solution prepared in the step (2) into the sample inlet of the chip treated in the step (3) to form a closed micro-pipeline/micro-cavity system in the chip, absorbing air in the micro-pipeline/micro-cavity system by using a degassing PDMS cover sheet to form negative pressure, and driving the aqueous phase liquid sample to enter and fill the micro-pipeline/micro-cavity system in the chip; simultaneously, silicone oil is dripped into the oil storage cavity of the cover plate layer to fill the oil storage cavity;
(4) Sample discretization: after the whole micro-pipeline/micro-cavity system in the chip is filled with the water phase liquid sample, peeling off the PDMS cover plate layer from one end close to the oil storage cavity, and driving the oil phase to be paved on the surface of the hydrophobic micro-structure layer due to the wettability of the oil phase liquid to the surface of the PDMS hydrophobic micro-structure layer and the capillary action of a contact gap between the cover plate layer and the structure layer which moves in the peeling process; meanwhile, based on the wettability difference of oil phase and water phase liquid on the hydrophobic surface of the hydrophobic microstructure layer and the surface tension effect of the water phase liquid, the water phase liquid in the micro-pipeline is automatically broken and pushed into the connected micro-cavities by the surface tension, so that a liquid sample is dispersed into each micro-cavity, and the discretization of the water phase sample liquid is realized.
(5) Sealing a liquid sample: after discretization of the aqueous phase sample liquid is completed, a piece of glass sealing cover of the spin-coating PDMS prepolymer is covered on the chip hydrophobic microstructure layer with the cover layer peeled off, so that sealing of the discretized liquid phase sample is realized, and the formed glass-PDMS-glass sandwich structure ensures low moisture volatilization in the incubation culture process of escherichia coli;
(6) And (3) incubation and amplification: placing the chip sealed in the step (5) in a constant-temperature incubator to incubate for 5 hours at 37 ℃;
(7) Signal acquisition and analysis: the fluorescence signal reading and data analysis were performed by fluorescence microscopy on the chip after the completion of the incubation in step (6), and the results are shown in FIG. 4, in which (a) to (d) of FIG. 4 show that the E.coli concentration in the liquid phase sample was 10, respectively 5 、10 4 、10 3 、10 2 Results of incubation amplification of CFU/. Mu.L.
Example 3:
a simple, convenient and rapid liquid sample discretization chip is applied to micro-nano particle synthesis, and is synthesized by polyethylene glycol diacrylate (PEGDA) gel microspheres as an example, and the specific steps are as follows:
(1) Chip preparation: manufacturing a PDMS hydrophobic microstructure layer containing 20000 micro-cavities and a communicated micro-pipeline network by combining a photoetching mold manufacturing process with a PDMS pouring reverse mold, wherein each micro-cavity is cylindrical, the diameter of each micro-cavity is 100 micrometers, and the depth of each micro-cavity is 100 micrometers; simultaneously, a 2 mm-thick chip PDMS cover plate layer comprising a sample inlet and an oil storage cavity is manufactured through PDMS pouring and punching; then, aligning and attaching the cover sheet layer and the hydrophobic microstructure layer, and assembling to obtain a liquid-phase rapid discretization chip; specific procedures for chip preparation reference example 1;
(2) Degassing the chip: placing the chip obtained in the step (1) in a vacuum container for degassing treatment for at least 30 minutes, and carrying out vacuum packaging for later use;
(3) Sample introduction: dripping 40 microliters of sample liquid (which is formed by mixing 1.6 microliters of 0.01 g/ml rhodamine B, 30 microliters of ethanol, 8 microliters of PEGDA and 0.4 microliters of 1173 photoinitiator (2-hydroxy-2-methyl-1-phenyl-1-propane, HMPP) into the sample inlet of the chip treated in the step (2) to form a closed micro-pipeline/micro-cavity system in the chip, absorbing air in the micro-pipeline/micro-cavity system by using a degassing PDMS cover plate to form negative pressure, driving the liquid phase sample to enter and fill the micro-pipeline/micro-cavity system in the chip, and simultaneously dripping n-hexadecane into an oil storage cavity of the cover plate layer to fill the oil storage cavity;
(4) Sample discretization: after the whole micro-pipeline/micro-cavity system in the chip is filled with the water phase liquid sample, peeling the PDMS cover sheet layer from one end close to the oil storage cavity, and driving the oil phase to be fully paved on the surface of the hydrophobic micro-structure layer due to the wettability of the oil phase liquid on the surface of the PDMS hydrophobic micro-structure layer and the capillary action of a contact gap between the cover sheet layer and the structure layer which moves in the peeling process; meanwhile, based on the wettability difference of oil phase and water phase liquid on the hydrophobic surface of the hydrophobic microstructure layer and the surface tension effect of the water phase liquid, the water phase liquid in the micro-pipeline is automatically broken and pushed into the connected micro-cavities by the surface tension, so that a liquid sample is dispersed into each micro-cavity, and the discretization of the water phase sample liquid is realized, and the result is shown in fig. 5 a;
(5) Spheroidizing a discretized sample: after the discretization of the aqueous phase sample liquid is completed, the chip is placed on a room temperature or a hot plate, so that ethanol in the discretized liquid drop is volatilized, the discretized aqueous phase liquid drop is contracted, and the aqueous phase liquid drop forms a spherical shape in the continuous oil phase under the action of surface tension, and the formed spherical liquid drop has good uniformity because all micro cavities have the same volume (as shown in fig. 5 b);
(6) Polymerizing and forming: after the discretization sample is spherical, exposing the chip to 330-380 nm ultraviolet light of 100W for 120 minutes, and polymerizing the spherical aqueous phase droplets to form PEGDA gel microspheres in a photocuring mode, wherein the results are shown in FIGS. 5c and 5d;
(7) And (3) microsphere collection: after the polymerization is completed, the chip is soaked in isopropanol for 5 minutes, shaken and finally centrifuged to collect PEGDA pellets.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (4)
1. A simple, convenient and fast discretization chip for liquid samples is characterized by comprising a cover layer and a hydrophobic microstructure layer, wherein the cover layer comprises at least one sample inlet and an oil storage cavity, and the hydrophobic microstructure layer comprises at least one microcavity array, a micro-pipeline array communicated with each microcavity in each microcavity array and at least one main sample inlet pipeline; the cross section of the micro-pipeline is smaller than the longitudinal cross section of the micro-cavity; the main sampling pipeline is positioned at one end of the microcavity array or positioned around the microcavity array, and the cross section of the main sampling pipeline is larger than that of the micro pipeline; the cover plate layer is a polydimethylsiloxane flat plate or a polyurethane flat plate, and the thickness of the cover plate layer is more than 1 mm; after the cover plate layer is attached and assembled with the hydrophobic microstructure layer, the oil storage cavity is not communicated with the microcavity array and the micro pipeline; the use method of the simple and rapid liquid sample discretization chip comprises the following steps:
(1) Preparing: aligning, fitting and assembling the cover plate layer and the hydrophobic microstructure layer, and placing the assembled chip in a vacuum container for degassing treatment;
(2) Filling a sample: taking out the chip subjected to degassing treatment in the step (1), and dropwise adding the aqueous phase liquid sample to be discretized at a sample inlet of the chip; meanwhile, oil phase liquid is dripped into the oil storage cavity;
(3) Sample discretization: and (3) after the chip sample filling in the step (2) is finished, peeling the cover sheet layer of the chip from one end close to the oil storage cavity from the hydrophobic microstructure layer, and realizing the discretization of the aqueous phase sample liquid.
2. The simple and fast discretization chip of liquid sample according to claim 1, wherein the microcavity array comprises a plurality of cylindrical microcavities, the cross section of each cylindrical microcavity is circular or regular polygon, and the geometric shapes and sizes of all the cylindrical microcavities in each microcavity array are identical.
3. The liquid sample simple and rapid discretization chip of claim 1, wherein the hydrophobic microstructure layer is made of silicon, glass, polymethyl methacrylate, polydimethylsiloxane, polycarbonate, polyethylene terephthalate, cyclic olefin copolymer, polystyrene or epoxy resin, or a composite structure material with polydimethylsiloxane or cyclic olefin copolymer as a structural surface and glass or silicon as a supporting substrate.
4. The simple and rapid discretization chip for liquid samples according to claim 1, wherein the degassing treatment time in step (1) is at least 30 minutes.
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