CN109395788B - Chip device for preparing liquid drops in tube - Google Patents
Chip device for preparing liquid drops in tube Download PDFInfo
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- CN109395788B CN109395788B CN201811430323.9A CN201811430323A CN109395788B CN 109395788 B CN109395788 B CN 109395788B CN 201811430323 A CN201811430323 A CN 201811430323A CN 109395788 B CN109395788 B CN 109395788B
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Classifications
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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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- 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
- B01L2200/10—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
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
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- Chemical Kinetics & Catalysis (AREA)
- Physical Or Chemical Processes And Apparatus (AREA)
Abstract
The device comprises a reaction tube and a droplet generation chip, wherein the droplet generation chip comprises a reagent inlet and a plurality of branch micro-channels which are connected with the reagent inlet and have consistent size structures, the branch micro-channels are parallel to the bottom of the droplet generation chip and are distributed in a way of diverging outwards from the center of the chip, the outlets of the branch micro-channels are positioned at the outer edge of the side wall of the droplet generation chip, a nozzle structure is formed for droplet preparation, the reaction tube is loaded with continuous phase reagent in the process of droplet preparation, the continuous phase reagent is fully submerged and infiltrates the branch micro-channels and the nozzle structure of the droplet generation chip in the whole droplet preparation process, and the disperse phase reagent is loaded through the reagent inlet and is broken to form droplets through the nozzle structure by the branch micro-channels under the action of pressure; the invention improves the device integration performance, simplifies the liquid drop generation process, reduces the cost and the complexity of an external pressure driving control system, improves the liquid drop preparation frequency to the maximum extent, and meets the analysis requirements of different application occasions.
Description
Technical Field
The invention belongs to the technical field of biochemical microfluidics, and particularly relates to a chip device for preparing in-tube liquid drops.
Background
In the field of biochemical microfluidic analysis for enzyme synthesis and activity analysis thereof, liquid drop digital PCR, cell high-throughput screening and the like, the liquid drop microfluidic technology has undergone rapid development, and micro liquid drops are often used as discrete biochemical reaction containers with nanoliter or picoliter or even femtoliter grades, so that the method has great advantages in the precision, sensitivity and repeatability of analysis and detection. In addition, the microfluidic technology has the advantages of automation, easy integration and high flux, and can also greatly improve the sensitivity and efficiency of biochemical analysis. Droplets are typically composed of a continuous phase and a dispersed phase of immiscible reagents, with the interaction of flow shear forces and interfacial tension between the two fluids separating the dispersed phase into discrete nanoliter and below volume microdroplet units.
With the development of microfluidic technology, microfluidic-based droplet preparation methods have been rapidly applied and developed, and common structures include T-type, flow focusing (US 9500664B 2), coaxial flow (CN 105771826B), and stepped emulsification (step emulsification) forms. The step emulsification method generally only needs to control a disperse phase to realize droplet preparation, and parallelization and integration of the device are easier to realize. However, the droplets prepared in this method tend to be easily aggregated near the droplet preparation unit, which affects the stable preparation of subsequent droplets, and a High throughput step emulsion chip device for glass substrates is described in document Macromolecular Chemistry & Physics, volume 218, 2017, page 1600472, alessandro Ofner et al, "High-Throughput Step Emulsification for the Production of Functional Materials Using a Glass Microfluidic Device", which solves the problem of aggregation of droplets in the droplet preparation unit by controlling the flow flushing of the continuous phase reagent to drive the droplets out of the droplet preparation unit, but this increases the complexity of the chip device, and the droplets still need to be transferred into a reaction tube for subsequent reaction after preparation. Chinese patent (publication No. CN 105413772B) provides a single/multi-component droplet preparation device based on integrated micro-channels, which includes a discrete phase input system and a droplet generation system, but the droplet preparation device described in the patent requires multiple components to be connected, and is difficult to achieve miniaturization of the device, and the droplet preparation unit is composed of a micro-channel plastic film matched with a discrete phase outlet joint formed by a clamping plate and a backing plate, so as to form a series of integrated micro-channels which are arranged in parallel and have downward outlets, and the droplet size is limited by the structure of the integrated micro-channels and is difficult to reduce, and is only suitable for droplet preparation in which the density of the discrete phase is greater than that of the continuous phase.
Therefore, the conventional step emulsion droplet preparation method has defects in device miniaturization and integration, and requires complicated fluidic devices and chip designs to achieve large-scale preparation of small droplets.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an in-tube droplet preparation chip device, which can simplify the droplet generation process, reduce the cost and complexity of an external pressure driving control system, furthest improve the droplet preparation frequency, reduce the droplet transfer step, directly complete the subsequent reaction analysis in a tube, ensure that the volume of a reagent system (particularly a disperse phase) is adjustable in a larger range, meet the analysis requirements of different application occasions, and promote the development and application of a droplet microfluidic technology in the field of biochemical analysis.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an in-tube droplet preparation chip device comprises a reaction tube 1 and a droplet generation chip 2 assembled with the reaction tube, wherein the droplet generation chip 2 comprises a reagent inlet 3 and a plurality of branch micro-channels 4 with consistent size structures connected with the reagent inlet 3, the branch micro-channels 4 are parallel to the bottom of the droplet generation chip 2 and are distributed in a way of diverging outwards from the center of the chip, and the outlets of the branch micro-channels 4 are positioned at the outer edge of the side wall of the droplet generation chip 2 and form a nozzle structure 5 for droplet preparation;
a gap exists between the nozzle structure 5 and the inner wall 7 of the reaction tube;
the reaction tube 1 is loaded with a continuous phase reagent 8 during droplet preparation, the continuous phase reagent 8 is fully submerged and infiltrates the branch micro-channel 4 and the nozzle structure 5 of the droplet generation chip 2 during the whole droplet preparation process, the disperse phase reagent 9 is loaded through the reagent inlet 3, and is broken to form a droplet 6 after reaching the nozzle structure 5 through the branch micro-channel 4 under the action of pressure, and the droplet 6 is quickly separated from the nozzle structure 5 due to the density difference of the continuous phase reagent 8 and the disperse phase reagent 9 after being formed.
The reagent inlet 3 of the droplet generation chip 2 is reserved with a liquid storage cavity structure 302, during droplet preparation, reagent is firstly transferred into the liquid storage cavity structure 302, and then continuously controlled forward pressure is applied to the reagent inlet 3 to complete fluid driving and droplet preparation.
The sealing of the branch micro-channels 4 is completed by a flat plate structure 10, the flat plate structure 10 is positioned at the bottom of the liquid drop generating chip 2, the flat plate structure 10 and the liquid drop generating chip 2 are processed into a whole in a bonding mode, and the branch micro-channels 4 are processed on any side structure of a bonding surface or on both side structures and are aligned to complete micro-channel sealing.
The branch micro-channel 4 is a direct current channel or a curve-shaped channel, the cross section of the micro-channel is circular, semicircular, rectangular or other polygons, and the length of the branch micro-channel 4 is 0.5-15 mm.
The nozzle structure 5 is round, rectangular or other polygonal; the size of the rectangle is 5-500 um wide and 2-50 um high.
All or only the branch micro-channels 4 and the nozzle structures 5 of the in-tube droplet preparation chip device are subjected to hydrophilic and hydrophobic surface treatment; methods of surface treatment include direct coating, chemical deposition and chemical reaction modification.
The liquid drop generating chip 2 and the reaction tube 1 are assembled and combined in a one-to-one mode, namely, the liquid drop generating chip 2 is assembled and used with the corresponding reaction tube 1 which is connected through a connecting plate structure in a single, 2, 4, 8, 24, 48 or 96 combined mode, and the preparation of liquid drops of single, 2, 4, 8, 24, 48 or 96 and more disperse phase reagents can be completed in parallel.
A method of operating an in-tube droplet preparation chip device, comprising the steps of:
firstly, preparing a continuous phase reagent 8 and a disperse phase reagent 9 for preparing liquid drops, taking out a reaction tube 1 and a liquid drop generating chip 2 and a rubber pad 12 for sealing the liquid drop generating chip 2;
secondly, fixing the reaction tube 1, and adding a continuous phase reagent 8 into the reaction tube 1 by using a pipette matched with a disposable suction head;
thirdly, the liquid drop generating chip 2 is put into the reaction tube 1 for assembly, and after standing, the continuous phase reagent 8 is fully submerged and infiltrated into the branch micro-flow channel 4 and the nozzle structure 5 of the liquid drop generating chip 2 under the action of capillary force;
fourthly, sucking the disperse phase reagent 9 by a pipette matched with a disposable suction head and loading the disperse phase reagent into a liquid storage cavity structure 302 of the liquid drop generating chip 2; if the preparation of liquid drops of a plurality of samples is carried out simultaneously, the disposable suction head is replaced and the sample adding is repeated;
fifthly, covering the upper surface of the liquid drop generating chip 2 with a rubber pad 12, fixing the rubber pad 12 and enabling a vent hole on the rubber pad 12 to be aligned with the center of the liquid storage cavity structure 302;
step six, loading the constant forward pressure on the reagent inlet 3, and ending the droplet preparation process by fixed time or monitoring the pressure mutation after the droplet preparation is completed; the disperse phase reagent 9 reaches the nozzle structure 5 through the branch micro-channel 4 under the pressure effect, and breaks to form liquid drops 6 under the combined action of hydrodynamic force, two-phase interfacial tension, gravity and buoyancy; due to the density difference between the dispersed phase reagent 9 and the continuous phase reagent 8, the droplets 6 will leave the spout structure 5.
The rubber pad 12 and the droplet generation chip 2 are removed to obtain emulsion prepared in the reaction tube 1, and subsequent reaction and analysis can be directly carried out in the reaction tube 1.
Compared with the existing liquid drop preparation method and device, the invention has the following advantages:
(1) According to the device for preparing the chip by the liquid drops in the pipe, the nozzle structure 5 for realizing liquid drop generation is positioned at the outer edge of the side wall of the liquid drop generating chip 2, the liquid drops 6 can be quickly separated from the nozzle structure 5 by combining the density difference of the continuous phase reagent 8 and the disperse phase reagent 9, the transfer of the liquid drops 6 is independent of the flow of the continuous phase reagent 8, the complexity of a control system is reduced, and the consumption of the continuous phase reagent 8 is reduced. Compared with the prior art, the preparation of the liquid drops can be completed when the density of the continuous phase is larger or smaller than that of the disperse phase reagent, the restrictions on the continuous phase reagent 8 and the disperse phase reagent 9 are less, and the application range of the liquid drop preparation chip device is enlarged.
(2) In the in-tube droplet preparation chip device, the diameter of the droplet 6 mainly depends on the microscopic dimensions of the branch micro-channel 4 and the nozzle structure 5, so that the dimension of the droplet 6 is more controllable. Meanwhile, the change of the diameter of the liquid drops can be realized by adjusting the microscopic dimensions of the branch micro-flow channel 4 and the nozzle structure 5, and the macroscopic structures such as the reaction tube 1, the reagent inlet 3 and the like and the system driving control components are basically not required to be adjusted, so that a set of system and a plurality of specifications of liquid drop micro-fluidic systems can be customized according to experimental requirements, the cost of analysis and detection can be effectively reduced, and the application range of the system is expanded.
(3) The device for preparing the liquid drops in the pipe can prepare the subsequent liquid drops only by loading a proper amount of continuous phase reagent 8 into the nozzle structure 5 effectively at one time in use, and the nozzle structure 5 is always immersed in the continuous phase reagent 8 along with the increase of the amount of the reagent in the pipe along with the preparation of the liquid drops, so that the preparation of the liquid drops in a large scale can be continuously carried out. Therefore, for the chip device with the same specification, the volume and the liquid drop number of the disperse phase reagent 9 can be adjusted in a larger range without increasing the consumption of the continuous phase reagent 8, the duty ratio of the disperse phase reagent system in the emulsion system can be improved, the emulsion preparation requirements in different biochemical analysis fields can be met, and the compatibility and the expandability are good.
(4) Compared with the prior art, the in-tube droplet preparation chip device provided by the invention has the advantages that the preparation of droplets is directly finished in the reaction tube, no reagent residue and dead volume exist, almost all disperse phase reagents 9 can generate droplets 6 with uniform size, the next reaction and analysis process can be directly carried out, the transfer steps of the droplets 6 are reduced, the fusion and loss of the droplets 6 are avoided, and the integration level and the working efficiency of the system are improved.
(5) The device for preparing the chip by the liquid drop in the tube has a simple and reliable structure, can be processed in batches by adopting a precise injection molding or silicon micromachining process, and the reaction tube 1 can be replaced by a commonly used centrifuge tube in a laboratory, so that the cost of the chip device can be effectively reduced, and the universality of a subsequent analysis platform is also ensured.
Drawings
FIG. 1 is a schematic three-dimensional structure of a centrifuge tube-based in-tube droplet preparation chip device in example 1 of the present invention.
FIG. 2-1 is a top view of a centrifuge tube based in-tube droplet preparation chip device in example 1 of the present invention; fig. 2-2 is a cross-sectional view taken along A-A of fig. 2-1.
Fig. 3 is a schematic three-dimensional structure of a droplet-generating chip and a partial enlarged view of a branch microchannel at the bottom of the chip in example 1 of the present invention.
FIG. 4-1 is a schematic diagram of the droplet preparation process of the chip device in example 1 of the present invention, in which the density of the dispersed phase reagent is smaller than that of the discrete phase reagent; fig. 4-2 is a schematic diagram of the droplet preparation process of the chip device in example 1 of the present invention, in which the dispersed phase reagent density is greater than the discrete phase reagent density.
FIG. 5 is a schematic diagram showing the three-dimensional structure of an in-tube droplet-preparing chip device in the form of an eight-tube in example 2.
FIG. 6-1 is a top view of an in-tube droplet-making chip device in the form of an octant tube in example 2 of the present invention; fig. 6-2 is a B-B cross-sectional view of fig. 6-1.
FIG. 7 is a flow chart of the operation of the in-line droplet preparation chip device of the present invention.
FIG. 8 is a plot of droplet diameter versus capillary number (Ca) for chip devices of different size structures.
FIG. 9-1 is a schematic illustration of an experiment of droplet preparation in a tube of the present invention when the density of the dispersed phase is less than the density of the continuous phase reagent; FIG. 9-2 is a schematic illustration of an experiment of droplet preparation in a tube of the present invention when the density of the dispersed phase is greater than the density of the continuous phase reagent.
FIG. 10 is an experimental observation effect of an in-tube droplet fabrication chip device of the present invention, wherein FIG. (a) is a bright field imaging before an in-tube droplet generation chip device experiment; fig. (b), (c) and (d) are microscopic views of the process of preparing droplets in the tube of the chip device.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples.
Example 1 referring to fig. 1, 2-2, 3, 4-1, and 4-2, an in-tube droplet preparation chip apparatus includes a reaction tube 1 and a droplet generation chip 2 assembled therewith, the reaction tube 1 employs a centrifuge tube or PCR tube commonly used in laboratories, which is matched with the droplet generation chip 2 to complete droplet generation, and also serves as a container for droplet collection and reaction, taking a centrifuge tube of 0.5mL as an example; the liquid drop generating chip 2 is of a stepped cylindrical structure, and is assembled with the reaction tube 1 for preparing liquid drops; the liquid drop generating chip 2 is provided with a plurality of branch micro-channels 4 with consistent size and structure, wherein the reagent inlet 3 is connected with the reagent inlet 3, the branch micro-channels 4 are parallel to the bottom of the liquid drop generating chip 2 and are radially distributed outwards from the center of the chip, and the outlets of the branch micro-channels 4 are positioned at the outer edge of the side wall of the liquid drop generating chip 2 and form a nozzle structure 5 for preparing liquid drops; the liquid drop generating chip 2 forms coaxial clearance fit with the reaction tube 1 by virtue of the boss structure 202 outside the chip, so that the positioning of the liquid drop generating chip 2 is ensured, and meanwhile, the distance between the nozzle structure 5 and the inner wall 7 of the reaction tube is ensured.
Referring to fig. 4-1 and 4-2, the droplet-generating chip 2 and the reaction tube 1 are assembled and combined in a one-to-one manner. After assembly, the droplet generation chip 2 goes deep into the reaction tube 1, and maintains a gap of 0.5-1 mm with the inner wall 7 of the reaction tube, so that the droplets 6 and the continuous phase reagent 8 are prevented from overflowing the reaction tube 1 when 100 μl of reagent is added at most.
Referring to fig. 3, the reagent inlet 3 comprises a reservoir structure 302 for storing reagents, and the reservoir structure 302 is a conical cavity for storing no more than 100 μl of reagents.
Referring to fig. 3, the bottom of the droplet generating chip 2 is a flat plate structure 10, and is integrally processed with the droplet generating chip 2 by thermal compression bonding or medium bonding, etc., for encapsulating the branched micro flow channel 4 at the bottom of the droplet generating chip, ensuring the size of the micro flow channel and forming a nozzle structure 5 for droplet generation.
The nozzle structure 5 is rectangular with the width of 15-150 mu m and the height of 5-25 mu m, and a gap exists between the nozzle structure and the inner wall 7 of the reaction tube so as to meet the requirements of generating liquid drops 6 and separating from the nozzle structure 5; the nozzle structure 5 is parallel to the bottom of the chip, and is always immersed in the continuous phase reagent 8 in the preparation process of the liquid drops, so that the liquid drops 6 can be ensured to be rapidly separated from the nozzle structure 5 due to density difference, and the monodisperse micro-liquid drops with the diameters ranging from 15 mu m to 150 mu m are prepared through the nozzle structure 5.
The material of the droplet generating chip 2 adopts thermosetting or thermoplastic high molecular polymers, such as Polydimethylsiloxane (PDMS), polycarbonate (PC), polymethyl methacrylate (PMMA), polypropylene (PP), cycloolefin copolymer (COC) and the like; or an inorganic material such as silicon (Si), glass, etc.
All or only the branch micro-channels 4 and the nozzle structure 5 of the in-tube liquid drop preparation chip device are subjected to hydrophilic and hydrophobic surface treatment, so that the contact angle of the disperse phase reagent 9 on the surface of the nozzle structure 5 is more than 90 degrees, the adhesion and residue of the disperse phase reagent 9 are avoided, the generation of liquid drops 6 is promoted, and the liquid drops are quickly separated from the nozzle structure 5; methods of surface treatment include direct coating, chemical deposition and chemical reaction modification.
The working principle of the embodiment is as follows:
referring to fig. 4-1 and 4-2, the reaction tube 1 is loaded with the continuous phase reagent 8 during droplet preparation, and the continuous phase reagent 8 sufficiently floods and wets the branched micro-channels 4 and the nozzle structures 5 of the droplet-generating chip 2; the disperse phase reagent 9 is loaded into the liquid storage cavity structure 302 through the reagent inlet 3 and is subjected to continuous control forward pressureThe branch micro-channel 4 reaches the nozzle structure 5 to be broken to form liquid drops 6; the continuous phase reagent 8 and the disperse phase reagent 9 are two reagents which are mutually insoluble, the density of the two reagents is different, and the density difference is 0.1-1.0 g/cm 3 The method comprises the steps of carrying out a first treatment on the surface of the According to the density relation between the continuous phase reagent 8 and the disperse phase reagent 9, the liquid drops 6 are lifted to the liquid level or sunk to the bottom of the reaction tube 1 in the continuous phase reagent 8; as shown in fig. 4-1, when the density of the dispersed phase reagent 9 is smaller than that of the continuous phase reagent 8, the droplets 6 rise to the continuous phase liquid level position after being generated; as shown in fig. 4-2, when the density of the dispersed phase reagent 9 is greater than that of the continuous phase reagent 8, the droplets 6 sink to the bottom of the tube after being generated, without affecting the subsequent droplet generation.
Example 2 referring to fig. 5, 6-1 and 6-2, an in-tube droplet-preparing chip device comprising a reaction tube 1 and a droplet-generating chip 2; in order to realize large-scale droplet preparation and chip device batch production, the reaction tube 1 is processed in an eight-joint tube mode through a first connecting plate structure 101, and the reaction tube 1 adopts a flat-bottom round-table-shaped tube so as to reduce the consumption of continuous phase reagents; meanwhile, the droplet generation chip 2 is also manufactured in an eight-row mode through the second connecting plate structure 201 and assembled with the reaction tube 1, so that 8 groups of droplet preparation of the disperse phase reagents 9 can be simultaneously carried out, and a distance exists between the bottom of the droplet generation chip 2 and the bottom of the reaction tube 1 for storing the droplets 6 or the continuous phase reagents 8.
The reaction tube 1 and the droplet generation chip 2 are not limited to the eight-tube type, and the droplet generation chip 2 may be assembled with the reaction tube 1 in the form of an 8-tube or 96-well plate or the like in a single, 2, 4, 8, 24, 48 or 96-well plate or the like in combination, and the droplet preparation of the single, 2, 4, 8, 24, 48 or 96-well plate or more dispersed phase reagents 9 may be completed in parallel.
Referring to fig. 4-1, 4-2, 5, and 7, a method of operating an in-tube droplet preparation chip device, comprising the steps of:
firstly, preparing a continuous phase reagent 8 and a disperse phase reagent 9 for preparing liquid drops, taking out a reaction tube 1 and a liquid drop generating chip 2 and a rubber pad 12 for sealing the liquid drop generating chip 2;
secondly, fixing the reaction tube 1, and adding a continuous phase reagent 8 into the reaction tube 1 by using a pipette matched with a disposable suction head;
thirdly, the liquid drop generating chip 2 is put into the reaction tube 1 for assembly, and after standing, the continuous phase reagent 8 is fully submerged and infiltrated into the branch micro-flow channel 4 and the nozzle structure 5 of the liquid drop generating chip 2 under the action of capillary force;
fourthly, sucking the disperse phase reagent 9 by a pipette matched with a disposable suction head and loading the disperse phase reagent into a liquid storage cavity structure 302 of the liquid drop generating chip 2; if the preparation of liquid drops of a plurality of samples is carried out simultaneously, the disposable suction head is replaced and the sample adding is repeated;
covering the upper surface of the liquid drop generating chip 2 with a rubber pad 12, fixing the rubber pad 12 and enabling a vent hole on the rubber pad 12 to be aligned with the center of the liquid storage cavity structure 302 so as to facilitate the airtight connection of a subsequent pneumatic driving device;
step six, loading the constant forward pressure on the reagent inlet 3, and ending the droplet preparation process by fixed time or monitoring the pressure mutation after the droplet preparation is completed; the disperse phase reagent 9 reaches the nozzle structure 5 through the branch micro-channel 4 under the pressure effect, and breaks to form liquid drops 6 under the combined action of hydrodynamic force, two-phase interfacial tension, gravity and buoyancy; due to the density difference of the disperse phase reagent 9 and the continuous phase reagent 8, the liquid drops 6 are separated from the nozzle structure 5, and the phenomenon that the liquid drops float to the liquid level or sink to the bottom of the reaction tube 1 is shown in the continuous phase reagent 8, and the nozzle structure 5 is always immersed in the continuous phase reagent 8, so that the preparation of the subsequent liquid drops 6 is not influenced.
After the sixth step is completed, a negative pressure is applied to the reagent inlet 3 of the droplet generation chip 2, and part of the surplus continuous phase reagent 8 is transferred out of the reaction tube 1 through the droplet generation chip 2, so that the volume of the whole reaction system is reduced on the premise of not influencing the emulsion system, and the efficiency of the subsequent reaction process is improved.
The rubber pad 12 and the droplet generation chip 2 are removed to obtain emulsion prepared in the reaction tube 1, and subsequent reaction and analysis can be directly carried out in the reaction tube 1.
Referring to fig. 8, fig. 8 is a graph of droplet diameter versus capillary number (Ca) prepared for 8 sets of differently sized branch microchannels 4, the dimensions (width x height) of the branch microchannels 4 being 26.6um x 10.5um, 47.4um x 10.5um, 56.0um x 10.5um, 69.4um x 10.5um, 42.8um x 14.3um, 62.4um x 14.3um, 77.7um x 14.3um, 100.7um x 14.3um, respectively, with droplet diameter D increasing as the size of the branch microchannels 4 increases; for a branched microchannel 4 of fixed size, the variation in diameter of the droplet is relatively small in the range of the number of capillaries (Ca) shown; droplets with diameters in the range of 35um to 70um can be prepared by the 8 groups of size micro-channels. By further adjusting the size of the branch micro-channel 4, the chip device can form monodisperse liquid drops with the diameter ranging from 5um to 500um and the volume ranging from about 65fL to 65nL, thereby meeting the liquid drop preparation requirements of different application occasions.
Referring to fig. 9-1 and 9-2, when the chip device of the present invention is used to prepare droplets, the dispersed phase reagent 9 enters the branched micro-channel 4 under pressure and undergoes a step emulsification at the nozzle structure 5, and the principle is as follows: under the fluid flow condition of low capillary number, the disperse phase reagent 9 contacts with the continuous phase reagent 8 at the nozzle structure 5 and gradually expands to form spherical bulges under the action of interfacial tension, the internal and external pressures of the fluid are balanced, and the interface profile evolves under quasi-static state; according to the young-Laplace equation, as the spherical protrusion grows, the Laplace pressure Δp=2γ/R inside thereof gradually decreases. However, the curvature of the dispersed phase reagent 8 in the branched microchannels 4 has a minimum value kx=1/r, limited by the structural dimensions of the branched microchannels 4; when the size of the spherical bulge is increased to r or more, the internal pressure of the dispersed phase reagent 8 in the branch micro-channel 4 and the internal pressure of the spherical bulge at the nozzle structure 5 are not balanced any more; under the action of Laplace pressure, the extra disperse phase reagent 8 is driven into the spherical protrusions and causes the disperse phase reagent 8 fluid in the branch micro-channels 4 to form necking, the necking is broken under the action of interfacial tension, and the spherical protrusions are separated to form liquid drops 6; due to the density difference of the disperse phase reagent 9 and the continuous phase reagent 8, the liquid drops 6 are separated from the nozzle structure 5 on the side wall of the chip, and the phenomenon that the liquid drops float to the liquid level or sink to the bottom of the reaction tube 1 is shown in the continuous phase reagent 8, so that the aggregation of the liquid drops at the nozzle structure 5 is avoided, and the subsequent liquid drop preparation is not influenced.
Referring to fig. 10, fig. 10 shows experimental test results of an in-tube droplet fabrication chip device, in which a droplet generation chip 2 of PDMS material was fabricated using a soft lithography process, a continuous phase reagent 8 was a fluorinated oil containing a surfactant, and a dispersed phase reagent 9 was a PCR buffer; wherein figure (a) shows an in-tube droplet-generating chip device prior to the experiment; figures (b), (c), (d) show the in-tube droplet preparation process of the chip device; the diagram (c) is a local amplification observation result of the chip device, and it can be seen that the disperse phase reagent 9 forms liquid drops 6 at the nozzle structure 5 rapidly through the branch micro-channels, the liquid drops 6 float upwards to be separated from the nozzle structure 5, the preparation of subsequent liquid drops is not affected, the preparation speed of the liquid drops is high, and the size is uniform; the graph (d) shows that after a period of time of the droplet preparation experiment, the water-in-oil (W/O) droplets 6 are gathered on the upper layer of the liquid surface of the oil phase due to the density relationship, so that the preparation of the droplets in the large-scale tube can be realized, and the effect is good.
Claims (5)
1. An in-tube droplet fabrication chip device, characterized by: the device comprises a reaction tube (1) and a liquid drop generating chip (2) assembled with the reaction tube, wherein the liquid drop generating chip (2) and the reaction tube (1) form coaxial clearance fit one by one and are penetrated into the reaction tube (1), the liquid drop generating chip (2) comprises a reagent inlet (3) and a plurality of branch micro-channels (4) which are connected with the reagent inlet (3) and have the same size structure, the branch micro-channels (4) are parallel to the bottom of the liquid drop generating chip (2) and are distributed in a divergent mode from the center of the chip outwards, and the outlets of the branch micro-channels (4) are positioned at the outer edge of the side wall of the liquid drop generating chip (2) and form a nozzle structure (5) for preparing liquid drops;
the nozzle structure (5) is rectangular with the width of 10-500 mu m and the height of 2-50 mu m, and a gap exists between the nozzle structure (5) and the inner wall (7) of the reaction tube so as to meet the requirements of generating liquid drops (6) and separating from the nozzle structure (5);
the reaction tube (1) is loaded with a continuous phase reagent (8) in the preparation of liquid drops, the continuous phase reagent (8) is fully submerged and infiltrates into a branch micro-channel (4) and a nozzle structure (5) of the liquid drop generating chip (2) in the whole preparation process of the liquid drops, the disperse phase reagent (9) is loaded through a reagent inlet (3), and is broken to form liquid drops (6) through the branch micro-channel (4) to the nozzle structure (5) under the action of pressure, and the liquid drops (6) are quickly separated from the nozzle structure (5) due to the density difference of the continuous phase reagent (8) and the disperse phase reagent (9) after the liquid drops (6) are formed;
the reagent inlet (3) of the liquid drop generating chip (2) is reserved with a liquid storage cavity structure (302), reagent is firstly transferred into the liquid storage cavity structure (302) when the liquid drops are prepared, and then continuously controlled forward pressure is applied to the reagent inlet (3) to complete fluid driving and liquid drop preparation;
the sealing of the branch micro-channels (4) is completed by a flat plate structure (10), the flat plate structure (10) is positioned at the bottom of the liquid drop generating chip (2), the flat plate structure (10) and the liquid drop generating chip (2) are processed into a whole in a bonding mode, and the branch micro-channels (4) are processed on any side structure of a bonding surface or on both side structures and are aligned to complete micro-channel sealing;
the branch micro-channel (4) is a direct current channel or a curved flow channel, the cross section of the micro-channel is rectangular, and the length of the branch micro-channel (4) is 0.5-15 mm.
2. An in-tube droplet fabrication chip device according to claim 1, wherein: all or only the branch micro-channels (4) and the nozzle structures (5) of the in-tube liquid drop preparation chip device are subjected to hydrophilic and hydrophobic surface treatment; methods of surface treatment include direct coating, chemical deposition and chemical reaction modification.
3. An in-tube droplet fabrication chip device according to claim 1, wherein: the liquid drop generating chip (2) and the reaction tube (1) are assembled and combined in a one-to-one mode, namely, the liquid drop generating chip (2) is assembled and used with the corresponding reaction tube (1) connected through a connecting plate structure in a single, 2, 4, 8, 24, 48 or 96 combined mode, and the preparation of liquid drops of single, 2, 4, 8, 24, 48 or 96 or more disperse phase reagents can be completed in parallel.
4. A method of operating a chip device using in-tube droplet fabrication as defined in claim 1, comprising the steps of:
firstly, preparing a continuous phase reagent (8) and a disperse phase reagent (9) for preparing liquid drops, taking out a reaction tube (1) and a liquid drop generating chip (2) and a rubber pad (12) for sealing the liquid drop generating chip (2);
secondly, fixing the reaction tube (1), and adding a continuous phase reagent (8) into the reaction tube (1) by using a pipette matched with a disposable suction head;
thirdly, the liquid drop generating chip (2) is put into a reaction tube (1) for assembly, and after standing, the continuous phase reagent (8) is fully submerged and infiltrated into a branch micro-channel (4) and a nozzle structure (5) of the liquid drop generating chip (2) under the action of capillary force;
step four, sucking the disperse phase reagent (9) by a pipette matched with a disposable suction head and loading the disperse phase reagent into a liquid storage cavity structure (302) of the liquid drop generating chip (2); if the preparation of liquid drops of a plurality of samples is carried out simultaneously, the disposable suction head is replaced and the sample adding is repeated;
covering the upper surface of the liquid drop generating chip (2) with a rubber pad (12), fixing the rubber pad (12) and enabling a vent hole on the rubber pad (12) to be aligned with the center of the liquid storage cavity structure (302);
step six, loading the constant forward pressure on the reagent inlet (3), and ending the droplet preparation process through fixed time or monitoring the pressure mutation after the droplet preparation is finished; the disperse phase reagent (9) reaches the nozzle structure (5) through the branch micro-channel (4) under the action of pressure, and breaks to form liquid drops (6) under the combined action of fluid power, two-phase interfacial tension, gravity and buoyancy; due to the density difference between the dispersed phase reagent (9) and the continuous phase reagent (8), the liquid droplets (6) will leave the spout structure (5).
5. The method of operating an in-tube droplet fabrication chip device of claim 4, wherein: the rubber pad (12) and the liquid drop generating chip (2) are removed, so that the emulsion prepared in the reaction tube (1) is obtained, and the subsequent reaction and analysis can be directly carried out in the reaction tube (1).
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| CN110813395B (en) * | 2019-11-29 | 2022-02-08 | 南通大学 | Suction head droplet generating device for rapidly preparing micro droplets |
| CN115335141A (en) * | 2020-04-01 | 2022-11-11 | 默克专利股份有限公司 | Emulsifying device |
| CN111957360A (en) * | 2020-09-07 | 2020-11-20 | 深圳市亚辉龙生物科技股份有限公司 | Droplet microfluidic chip and preparation method of micro-droplets |
| CN112619719B (en) * | 2020-12-04 | 2022-03-29 | 深圳先进技术研究院 | Droplet generation microdevice for digital PCR |
| CN115518703A (en) * | 2021-06-24 | 2022-12-27 | 北京致雨生物科技有限公司 | Droplet generation device, system and method for generating droplets |
| CN113956968B (en) * | 2021-10-20 | 2023-11-14 | 西安天隆科技有限公司 | Liquid drop type digital PCR system and analysis method for realizing absolute quantification thereof |
| CN115155676A (en) * | 2022-02-18 | 2022-10-11 | 广东永诺医疗科技有限公司 | Single-pressure multi-channel droplet preparation chip |
| CN115228525A (en) * | 2022-06-30 | 2022-10-25 | 晶准生物医学（深圳）有限公司 | Liquid droplet generating gun head and liquid droplet generating device |
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