CN114752479A - Method and apparatus for generating liquid droplet - Google Patents

Abstract

The invention relates to a novel liquid drop generating method and a novel liquid drop generating device, which can realize stable co-flow injection of two mutually insoluble liquids through a simple first cavity, a simple second cavity and a simple hole structure, simultaneously, the inner layer liquid injection can be stably broken into uniform micro-droplets through reciprocating vibration of the first cavity and the second cavity, a micro-channel structure with micron magnitude is not needed in the whole generating process, the generating volume and the generating speed of the micro-droplets can be adjusted by controlling the vibration amplitude, the frequency and the liquid flow pushing speed, and the flux is high. Meanwhile, the device of the invention has simple structure, few parts and is easy to manufacture industrially; the method of the present invention can stably obtain uniform size liquid drops for biochemical samples, the volume and the generation rate of the liquid drops can be freely adjusted, and the method is not easily influenced by external force interference and is easy to implement industrially.

Description

Method and apparatus for generating liquid droplet

Technical Field

The invention relates to a droplet generation method and a device which are particularly suitable for the fields of digital PCR, single cell capture, droplet sorting, drug delivery and the like.

Background

PCR, Polymerase Chain Reaction (PCR), is a technique that can rapidly amplify a specific gene or DNA sequence in vitro. This technology is based on some characteristics of the DNA sequence in vivo which can be rapidly replicated, and realizes the rapid amplification of specific DNA sequence in vitro, and millions of copies of specific DNA sequence can be obtained in a short time. The development of PCR technology has gone through 3 generations of innovation so far. The first generation PCR adopts a common PCR instrument to amplify the target gene, and adopts agarose gel electrophoresis to analyze the product; the second generation PCR is a fluorescent quantitative PCR technology (Real-Time PCR, qPCR) which is used for monitoring the accumulation of an amplification product in Real Time by adding a fluorescent reagent capable of indicating the reaction process into a reaction system and quantifying the concentration of an initial target gene by means of the Ct value of a fluorescence curve; the third generation PCR technology, Digital PCR (Digital PCR, dPCR, Dig-PCR), is a novel method for detecting and quantifying nucleic acid, and can determine the absolute number of target molecules to be detected as low as a single copy by directly counting the target molecules without depending on any calibrator or external standard. The strategy of digital PCR technology can be referred to as "divide and conquer": the sample is diluted and divided into thousands of micro-reaction chambers, such that each reaction chamber contains only zero or one target gene sequence (a few contain more). By counting the number of reaction chambers with positive results, the researcher can know the absolute number of target gene molecules to be detected in the original sample. Because the digital PCR only judges whether two amplification states exist or not when the result is judged, the intersection point of a fluorescence signal and a set threshold line does not need to be detected, and the identification of a Ct value is not relied on completely, the influence of the amplification efficiency on the reaction of the digital PCR is greatly reduced, and the tolerance capability on PCR reaction inhibitors is greatly improved; the process of standard reaction system allocation in digital PCR experiments can greatly reduce the background sequence concentration which has a competitive effect with a target sequence, so that the digital PCR technology is particularly suitable for detecting rare mutation in a complex background. Compared with the traditional PCR technology, the digital PCR has the advantages of small sample quantity, reaction reagent saving, realization of absolute quantification of nucleic acid molecules, reduction of mutual interference between copies in the same sample, more excellent sensitivity and specificity and the like.

At present, there are two main types of digital PCR products on the market: microfluidic chip-based digital PCR and droplet-based digital PCR. The principle of microfluidic chip-based digital PCR technology is based on the fact that microfluidic devices can enable digital PCR to perform highly parallel analysis in one PCR reaction. Multilayer Soft Lithography (MSL) technology gives the ability to design integrated microfluidic circuits and control the cost-effectiveness of producing microfluidic chips. In principle, any molecular reaction that can be carried out in a reaction tube can be realized in a microfluidic chip. The ability to perform thousands of individual PCR reactions on a single MSL chip reduces reagent consumption and pipette steps necessary to break down individual samples into a large number of individual micro-wells (microwells) using microfluidic control valves and perform digital PCR in an automated fashion. In a microfluidic chip, a positive reaction contains at least one target molecule, and the number of positives in a very dilute sample is very similar to the number of targets. In case a positive reaction contains more than one target molecule in a sample with a higher concentration, even if a large proportion of the reactions are positive, they can be calculated by an algorithm based on the poisson distribution. The basic working principle of the droplet digital PCR technology is as follows: generating an aqueous phase microdroplet mixed sample in a certain way and manipulating it to move along a microchannel in an inert oil phase carrier solution; the microdroplets are not interfered with each other, and each microdroplet is an independent PCR microreactor; the microdroplets are typically much smaller in volume than the wells of a microtiter plate (widely used in first and second generation PCR techniques, such as 96-well plates) and can even be as small as a single cell assay. The data analysis method is similar to chip-based digital PCR. Since there are many techniques for generating and manipulating droplets, which gives the technology of microdroplet PCR a high degree of flexibility, researchers can even design individualized experimental protocols for each droplet.

Droplet generation technology based on Microfluidics (Microfluidics) has been rapidly developed in recent years, and droplet generation is based on interfacial instability of a dispersed phase when a continuous phase meets in a microchannel. Through different micro-fluidic channel chip designs, droplets with tiny volumes can be generated, and operations such as fusion, reaction, sorting and the like are performed; however, the generation of droplets on a chip needs to satisfy specific flow rate, oil-water interfacial tension, and conditions of channel configuration and channel surface modification, and the range of volume adjustment of droplets is also limited by the above factors. In addition, after the droplets are generated in the channel, the droplets need to be transferred to a storage container by a special step and device, conditions of the single droplets are difficult to customize, and positioning, extraction and analysis operations of the droplets are inconvenient. Another technique injects or jets a minute amount of liquid through a capillary or microchannel and injects the liquid into a micro-pit or spots on a substrate. This technique is in principle a simple drop generation strategy. However, in practice, when the liquid drop leaves the capillary tube, the surface tension of the liquid drop separating from the continuous liquid in the tube and the adhesion force of the liquid drop to the surface of the tube mouth exist, so that the volume quantification of the liquid drop is influenced. The kinetic energy of the liquid drop separated from the outlet is increased by adopting special jetting or liquid drop excitation modes such as piezoelectric ceramics, thermal expansion, ultrasound and the like so as to overcome the influence of surface tension, and the adhesive force of the liquid drop on the pipe orifice is reduced through the special configuration of the outlet of the micro-channel and silanization or coating treatment. However, these methods rely on more complicated fluid driving devices, are more expensive, and are difficult to accurately control the volume of the droplets. In addition, the method of directly aligning the outlet of the pipeline with the substrate or spotting the liquid in the micro-pits can be adopted, and the adhesive force of the liquid drop on the pipe orifice can be overcome by utilizing the contact adhesive force and the interfacial tension of the liquid drop and the substrate. However, for biochemical samples with complex components and different viscosity characteristics, a generally applicable method is still lacking, which can completely overcome the surface tension of the liquid attached to the microchannel port and avoid the residue of the liquid drop at the channel port and the volume error, nozzle blockage and cross contamination caused thereby.

In summary, the micro-reaction chamber generation technology (whether micro-fluidic digital PCR or micro-drop digital PCR) adopted by the current digital PCR product needs to perform complex and precise flow rate control on micro-flow or micro-drop, and needs to design a complex micro-flow channel to implement the operation of the experiment. Thus, the consumables of the digital PCR product are incompatible with the 96-well plate of qPCR and are expensive. There is a need in the market for digital PCR products based on simpler droplet generation and control techniques.

It is also known that, with the progress of research on cell physiology, scientists begin to analyze life phenomena at a single cell or single molecule level. There is a difference between cells for multicellular organisms, i.e., heterogeneity of genetic information. For example, in tumor tissue, the genetic information of the genome and transcriptome of cells in the center of the tumor and cells surrounding the tumor are different, and this difference clinically determines whether the tumor is effective for a particular therapy. The current technology for realizing single cell sequencing mainly comprises a micromanipulation technology; laser capture microdissection; flow cytometry sorting by laser-induced fluorescence detection; and microfluidic sorting, which is considered to be a comparatively ideal single-cell separation and analysis means. However, similar to the problems encountered in the generation of droplets by digital PCR, the microfluidic chip for capturing single cells also has the disadvantages of complicated design, processing and operation, high cost, difficulty in accurate quantification, limitation of the adjustment range due to many factors (such as specific flow rate, oil-water interfacial tension, channel configuration, channel surface modification, etc., and the influence of various hydrodynamics and channel structures on the volume of generated droplets), and the like.

Disclosure of Invention

The invention provides a novel liquid drop generation method and a liquid drop generation device, aiming at the existing problems of digital PCR liquid drop generation, single-cell liquid drop preparation and the like.

To solve the above problems, in one aspect, the present invention provides a droplet generation method particularly suitable for, but not limited to, preparing droplets of digital PCR, single cell droplets, by mixing a first liquid and a second liquid immiscible with the first liquid to form droplets, characterized in that the droplet generation method comprises the steps of:

inputting the first liquid into a first cavity with a liquid drop output port;

delivering said second liquid toward said drop delivery outlet via a second chamber at least partially inserted into said first chamber and having a liquid inlet and outlet, wherein said first liquid is used to drive the flow of said second liquid;

driving the first cavity and the second cavity to vibrate while inputting the first liquid and outputting the second liquid;

and the second liquid is wrapped by the first liquid in the first cavity after flowing out of the second cavity and is jetted out of the liquid drop output port together to obtain the liquid drop, wherein the first liquid is continuous phase liquid, and the second liquid is dispersed phase liquid.

Preferably, the vibration is reciprocating vibration, and the direction of the reciprocating vibration is perpendicular to the center line direction of the liquid inlet and outlet.

Preferably, the liquid inlet and outlet of the second cavity and the central line of the liquid drop outlet of the first cavity extend along the vertical direction, the liquid inlet and outlet is located right above the liquid drop outlet, and the reciprocating vibration is reciprocating vibration along the horizontal direction.

Preferably, the amplitude of the vibration is 5-500 microns, and/or the frequency of the vibration is 10HZ-10 KHZ.

Preferably, the first liquid and the second liquid are respectively driven to flow by pumps, wherein the speed of inputting the first liquid is 1000 ml/h-10000 ml/h, and the speed of outputting the second liquid is 1 ml/h-100 ml/h.

Preferably, in the process of generating the liquid droplets, the first cavity and the second cavity are synchronously vibrated, and the pump for driving the first liquid and the second liquid independently and selectively keeps synchronously vibrating with the first cavity and the second cavity or does not vibrate.

Preferably, the first liquid is an oil phase and the second liquid is an aqueous phase containing the biological or chemical substance to be detected.

Preferably, the second cavity is a single cavity or a multi-cavity, the multi-cavity is 2, 3, 4 or more cavities, and the multiple cavities are concentrically arranged or arranged side by side or partially concentrically arranged side by side.

Further preferably, the second cavity is a single cavity, the first liquid is filled in the second cavity, the second liquid is sucked into the second cavity in which the first liquid is stored through the liquid inlet and outlet, and the first liquid is driven to drive the second liquid to be output from the second cavity.

Further preferably, the second cavity is a single cavity, the second liquid is formed by driving a plurality of liquids to mix before entering the second cavity or after mixing in the second cavity, and each of the plurality of liquids is driven by the first liquid.

Further preferably, the second cavity be the multi-chamber, include outer cavity and be located outer cavity in and rather than the concentric inner chamber that sets up, the second liquid include first component and second component, the branch road of saying of the liquid of outer cavity and inner chamber export first component and second component, export first component, second component both by first liquid parcel in the first cavity and spray jointly the drop delivery outlet, obtain the drop.

Further preferably, the second chamber is a multi-chamber, and comprises a plurality of sub-chambers arranged side by side, the second liquid correspondingly comprises a plurality of sub-components, a plurality of sub-components are respectively output through the plurality of sub-chambers one by one, and the output plurality of sub-components are wrapped by the first liquid in the first chamber and are jointly ejected out of the liquid drop output port to obtain the liquid drop.

The invention provides a liquid drop generating device, which comprises a vibrating device for outputting vibration, a first chamber for generating liquid drops and a second chamber, wherein the first chamber comprises a first cavity, a first liquid inlet and a liquid drop outlet which are communicated with the first cavity, the second chamber comprises a second cavity, a liquid inlet and a liquid outlet which are communicated with the second cavity and are used for the inlet and the outlet of the second liquid, and the vibrating device is connected with the first chamber and is used for driving the first chamber to vibrate integrally.

Preferably, the vibration device is used for driving the first chamber to vibrate in a reciprocating manner in the horizontal direction.

Preferably, the lower part of the second chamber is inserted into the first cavity, and the liquid inlet and outlet are formed in the lower part of the second chamber, and the liquid inlet and outlet of the second chamber are located right above the liquid drop outlet of the first chamber.

Further preferably, a gap of 0.1-5 mm is formed between the liquid drop outlet of the first chamber and the liquid inlet and outlet at one end of the second chamber.

Preferably, the liquid drop generating device further comprises a vibration transmission cover, the vibration device is connected with the vibration transmission cover, and the vibration transmission cover is sleeved outside the first chamber.

Preferably, the droplet generating device further comprises a pump for providing the driving force required for each liquid flow.

Further preferably, the pump may be connected to the first and second chambers by a flexible connection.

Preferably, the first chamber is detachably connected to the second chamber, the first chamber includes an interface connected to the first chamber, and the second chamber is disposed through the interface. Further preferably, a sealing structure is arranged at the interface.

Preferably, the second chamber includes a tube body having openings at both ends, the openings at both ends respectively forming the liquid inlet and outlet, and the lower portion of the tube body being narrowed near the openings to form a relatively narrower liquid inlet and outlet than other portions of the tube body.

Further preferably, the second chamber further comprises one, two or more connections for connecting to other pipes disposed on the pipe, the droplet generating apparatus further optionally comprises one or more inlet pipes connected to the pipe through the connections, and the second chamber and the one or more inlet pipes are used for respectively introducing different components of the second liquid.

Further preferably, the tube body comprises an outer tube body and an inner tube body which is positioned in the outer tube body and is concentrically arranged with the outer tube body, the liquid inlet and outlet at the lower end of the inner tube body is positioned above the liquid inlet and outlet at the lower end of the outer tube body, and the outer tube body and the inner tube body are used for respectively introducing different components of the second liquid.

Further preferably, the tube body comprises a plurality of at least partially side-by-side arranged sub-tube bodies for respectively introducing different components of the second liquid.

Preferably, the vibration device may employ various high-frequency vibration generators conventional in the art, such as high-frequency mechanical vibration generators (piezo ceramic devices, voice coil motors, MEMS, etc.).

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

the invention can realize stable co-current injection of two immiscible liquids through the first cavity, the second cavity and the hole structure, and simultaneously, the inner layer liquid injection can be stably broken into even micro-droplets through carrying out reciprocating vibration modulation on the first cavity and the second cavity, and the whole generation process does not need a micro-channel structure with micron order and has high flux.

The device of the invention has simple structure, few parts and is easy to manufacture industrially; the method of the present invention can stably obtain uniform size liquid drops for biochemical samples, the volume and the generation rate of the liquid drops can be freely adjusted, and the method is not easily influenced by external force interference and is easy to implement industrially.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is an exploded view of a droplet generator according to one embodiment;

FIG. 2 is a schematic view of a droplet generating apparatus according to a first embodiment;

FIG. 3 is a schematic view showing the structure of a second chamber in the second embodiment;

FIG. 4 is a schematic structural view of a second chamber in the third embodiment;

FIG. 5 is a schematic view showing the structure of a second chamber in the fourth embodiment.

In the above drawings:

1. a first chamber; 10. a first cavity; 10a, a first liquid inlet; 10b, a droplet outlet; 10c, a valve; 10d, an interface;

2. A second chamber; 20. a second cavity; 20a, 20b, liquid inlet and outlet; 20c, a connecting part; 210. an outer tubular body; 211. an inner tube body; 22. a pipe dividing body;

3. a vibrating device; 30. a vibration transmission housing;

4a, 4b, a pump; 5. a collector; 6. a liquid inlet pipeline.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in a specific case to those of ordinary skill in the art.

The first embodiment is as follows:

a droplet generator as shown in figures 1 and 2 comprises a first chamber 1, a second chamber 2, a vibration device 3, pumps 4a, 4b, a collector 5, etc.

The first chamber 1 includes a first cavity 10, a first liquid inlet 10a communicating with the first cavity 10, and a liquid drop outlet 10b, the first liquid inlet 10a is used for connecting with the pump 4a, and a valve 10c is disposed at the liquid drop outlet 10b, and the inner diameter thereof may be generally 0.1-5 mm. Furthermore, the first chamber 1 comprises a port 10d for connection to the second chamber 2, where a resilient sealing ring or other sealing means is preferably arranged at the port 10 d. In this embodiment: the interface 10d and the droplet discharge port 10b are respectively located at the upper and lower sides of the first chamber 1.

The second chamber 2 includes a second chamber 20, and liquid inlets and outlets 20a and 20b communicating with the second chamber 20 for the inlet and outlet of the second liquid. In this embodiment: the second chamber 2 is a tube body with openings at two ends, the openings at two ends form liquid inlets and outlets 20a and 20b respectively, the liquid inlet and outlet 20a at one end of the tube body is used for being connected with the pump 4b, the other end of the tube body is narrowed near the opening to form a liquid inlet and outlet 20b which is relatively narrower than other parts of the tube body, and the inner diameter of the liquid inlet and outlet 20b can be usually 0.1-5 mm.

Connected to the first chamber 1 is a vibration device 3 which may be any of a variety of high frequency vibration generators conventional in the art, such as high frequency mechanical vibration generators (piezo ceramic devices, MEMS, etc.). In this embodiment: the device further comprises a vibration transmission cover 30, the vibration device 3 is connected with the vibration transmission cover 30, the vibration transmission cover 30 is sleeved outside the first chamber 1, the vibration device 3 applies reciprocating vibration to the first chamber 1 through the vibration transmission cover 30, and then the vibration device drives the second chamber 2 inserted into the first chamber 1 to perform reciprocating vibration together.

Further, the pumps 4a and 4b may be plunger pumps or the like.

The first chamber 1, the second chamber 3, the vibration means 3 and the pumps 4a, 4b may all be separate components, connected in use.

When in use: the second chamber 2 is vertically inserted into the first cavity 10 through the interface 10d, the connection between the first chamber and the second chamber can be kept sealed and stable by the elastic sealing ring at the interface 10d, the liquid inlet and outlet 20b at the lower end of the second chamber 2 is positioned right above the liquid drop outlet 10b of the first chamber 1, and a gap of 0.1-5 mm is formed between the liquid drop outlet 10b of the first chamber 1 and the liquid inlet and outlet 20b at one end of the second chamber 2.

The pump 4a is connected with the first liquid inlet 10a of the first chamber 1, and the pump 4b is connected with the liquid inlet and outlet 20a of the second chamber 2, specifically: the pumps 4a, 4b and the first liquid inlet 10a and the liquid outlet 20a can be connected by flexible connecting members, so that the pumps 4a, 4b are always fixed when the vibration device 3 drives the first chamber 1 and the second chamber 2 to perform reciprocating vibration.

The preparation of digital PCR droplets can be carried out by using the droplet generating device, and the process is as follows:

the second chamber 2 can be used to suck the continuous phase liquid (oil phase) A through the liquid inlet and outlet 20B by the control of the pump 4B, and continuously suck the dispersed phase liquid (water phase) B through the liquid inlet and outlet 20B, the volume of the sucked dispersed phase liquid does not exceed the volume of the second cavity 20, and the dispersed phase liquid A and the liquid B are not mutually soluble. The second chamber 2 is inserted into the first chamber 10 until its liquid inlet/outlet 20b is close to the droplet outlet 10 b.

Then, the first chamber 11 is filled with the continuous phase liquid C, the valve 10C is opened, and the pump 5a and the pump 5B are pushed at the same time, so that the dispersed phase liquid B in the second chamber 20 and the continuous phase liquid C in the first chamber 10 co-flow to be ejected out of the droplet outlet 10B. The driving speed of the pump 5a is 1-100 ml/h, and the driving speed of the pump 5b is 1000-10000 ml/h. While the double pumps are pushed, the vibration device 4 carries out high-frequency mechanical reciprocating vibration with the amplitude of 5-500 micrometers and the frequency of 10HZ-10KHZ, the first cavity 10 and the second cavity 20 are driven to make the first cavity 10 and the second cavity 20 generate reciprocating vibration in the left-right direction of the figure, and the jet flows of the dispersed phase liquid B and the continuous phase liquid C are simultaneously broken into uniform droplets according to the vibration frequency of the vibration device 4 and flow into the collector 6 together. In the collector 6, the continuous phase liquid is recombined into a complete oil phase, while the dispersed phase liquid B remains uniformly dispersed.

When the dispersed phase liquid B completely flows into the collector 6, the whole process is finished. The second chamber 2 is pulled out of the first cavity 10, and the second chamber 2 can be directly used for preparing the next PCR liquid drop or the next PCR liquid drop after being replaced by a new second chamber 2.

With the above droplet generating method, the generation position, the generation volume, the generation rate, and the composition of droplets of the droplets can be easily controlled. In particular, the length and thickness of the jet formed by the ejection of the second liquid from the drop outlet may be adjusted by adjusting one or more of the vibration frequency, vibration amplitude, relative rates of different liquid flows, viscosity of the liquid, etc., to obtain drops of a desired volume and desired composition at a desired location and at a desired rate, respectively.

The second embodiment:

the present embodiment is substantially the same as the first embodiment, except for the specific structure of the second chamber 2:

as shown in fig. 3: the second chamber 2 also comprises one, two or more connections 20c provided on the tubular body for connecting further conduits. The droplet generating apparatus may further optionally comprise one or more liquid inlet pipes 6 connected to the pipe body via a connecting portion 20c, the second chamber 2 and the one or more liquid inlet pipes 6 for respectively introducing different components of the second liquid, wherein the connecting portion 20c and the liquid inlet pipes 6 are provided in two sets in the figure. The second liquid is formed by driving a plurality of liquids to mix either before entering the second chamber 20 or within the second chamber 20, each of the plurality of liquids being driven by the first liquid.

Example three:

the present embodiment is substantially the same as the first embodiment, except for the specific structure of the second chamber 2:

as shown in fig. 4: the tube body of the second chamber 2 comprises an outer tube body 210 and an inner tube body 211 which is positioned in the outer tube body 210 and is concentrically arranged with the outer tube body, an outer cavity is formed in the outer tube body 210, an inner cavity is formed in the inner tube body 211, and the liquid inlet and outlet 20b 'at the lower end of the inner tube body 211 is positioned above the liquid inlet and outlet 20 b' at the lower end of the outer tube body 210. In this embodiment: the second liquid comprises a first component and a second component, the outer tube body 210 and the inner tube body 211 are used for respectively introducing different components of the second liquid, the first component and the second component are respectively output through liquid inlets and outlets 20b 'and 20 b' of the outer cavity of the outer tube body 210 and the inner cavity of the inner tube body 211, the first component and the second component are respectively driven by the first liquid, and the first component and the second component are output, wrapped by the first liquid in the first cavity 10 and jointly ejected out of a liquid drop output port 10b, so that liquid drops are obtained.

Example four:

this embodiment is substantially the same as the first embodiment, except for the specific structure of the second chamber 2:

as shown in fig. 5: the body of second room 2 includes a plurality of at least parts branch body 22 that set up side by side, forms the cavity in every branch body 22, and a plurality of branch bodies 22 are used for letting in the different component parts of second liquid respectively. In this embodiment: taking two as an example, the second liquid includes a first component and a second component, the first component and the second component are respectively output through two sub-cavities, the first component and the second component are respectively driven by the first liquid, and the first component and the second component are output to be wrapped by the first liquid in the first cavity 10 and jointly eject out the liquid drop output port 10b to obtain a liquid drop.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (26)

1. A droplet generation method suitable for preparing digital PCR droplets, single cell droplets, by mixing a first liquid and a second liquid immiscible with the first liquid to form droplets, characterized in that the droplet generation method comprises the following steps:

Inputting the first liquid into a first cavity with a liquid drop output port;

delivering said second liquid toward said drop delivery outlet via a second chamber at least partially inserted into said first chamber and having a liquid inlet and outlet, wherein said first liquid is used to drive the flow of said second liquid;

driving the first cavity and the second cavity to vibrate while inputting the first liquid and outputting the second liquid;

and the second liquid is wrapped by the first liquid in the first cavity after flowing out of the second cavity and is sprayed out of the liquid drop output port together to obtain the liquid drop, wherein the first liquid is continuous phase liquid, and the second liquid is dispersed phase liquid.

2. A droplet generation method according to claim 1, characterized in that: the vibration is reciprocating vibration, and the direction of the reciprocating vibration is vertical to the direction of the central line of the liquid inlet and the liquid outlet.

3. A droplet generation method according to claim 1, characterized in that: the liquid inlet and outlet of the second cavity and the central line of the liquid drop output port of the first cavity extend along the vertical direction, the liquid inlet and outlet are positioned right above the liquid drop output port, and the reciprocating vibration is the reciprocating vibration along the horizontal direction.

4. A droplet generation method according to claim 1, characterized in that: the vibration amplitude is 5-500 micrometers, and/or the vibration frequency is 10HZ-10 KHZ.

5. A liquid droplet generation method according to claim 1, characterized in that: the first liquid and the second liquid are driven to flow by pumps respectively, wherein the speed of inputting the first liquid is 1000 ml/h-10000 ml/h, and the speed of outputting the second liquid is 1 ml/h-100 ml/h.

6. A liquid droplet generation method according to claim 1, characterized in that: in the process of generating the liquid drops, the first cavity and the second cavity are synchronously vibrated, and the pumps for driving the first liquid and the second liquid independently and selectively keep synchronous vibration with the first cavity and the second cavity or do not vibrate.

7. A liquid droplet generation method according to claim 1, characterized in that: the first liquid is an oil phase and the second liquid is an aqueous phase containing the biological or chemical substance to be detected.

8. A liquid droplet generation method according to claim 1, characterized in that: the second cavity be single cavity or multi-chamber, the multi-chamber be 2, 3, 4 or more cavitys, concentric setting or side by side setting or partial concentric part is set up side by side between a plurality of cavitys.

9. A droplet generation method according to claim 8, wherein: the second cavity is a single cavity, the first liquid is filled in the second cavity, then the second liquid is sucked into the second cavity in which the first liquid is stored through the liquid inlet and the liquid outlet, and finally the first liquid is driven to drive the second liquid to be output from the second cavity.

10. A droplet generation method according to claim 8, wherein: the second cavity is a single cavity, the second liquid is formed by driving a plurality of strands of liquid to be mixed before entering the second cavity or mixed in the second cavity, and each strand of the plurality of strands of liquid is driven by the first liquid respectively.

11. A droplet generation method according to claim 8, wherein: the second cavity be the multi-chamber, include outer cavity and be located outer cavity in and rather than the concentric interior cavity that sets up, the second liquid include first component and second component, pass through respectively the liquid import and export output of outer cavity and interior cavity first component and second component, the output first component, second component two quilt first liquid parcel in the first cavity and spray jointly the liquid drop delivery outlet, obtain the liquid drop.

12. A droplet generation method according to claim 8, wherein: the second cavity is a multi-cavity body and comprises a plurality of sub-cavity bodies arranged side by side, the second liquid correspondingly comprises a plurality of sub-components which are respectively output in a plurality of sub-cavity bodies one by one, the output sub-components are wrapped by the first liquid in the first cavity body and jointly ejected to the liquid drop output port, and the liquid drops are obtained.

13. A droplet generation device, suitable for preparing digital PCR droplets, single cell droplets, the device including a vibration device for outputting vibrations, a first chamber for droplet generation, and a second chamber, characterized in that: the first chamber comprises a first cavity, a first liquid inlet and a liquid drop outlet, the first liquid inlet is communicated with the first cavity, the liquid drop outlet is communicated with the second cavity, the second chamber comprises a second cavity, a liquid inlet and a liquid outlet, the liquid inlet and the liquid outlet are communicated with the second cavity and used for the second liquid to enter and exit, and the vibration device is connected with the first chamber and used for driving the first chamber to vibrate integrally.

14. A droplet generator according to claim 13, wherein: the vibration device is used for driving the first chamber to vibrate in a reciprocating mode in the horizontal direction.

15. A droplet generator according to claim 13, wherein: the lower part of the second chamber is inserted into the first cavity, the lower part of the second chamber is provided with the liquid inlet and outlet, and the liquid inlet and outlet of the second chamber are positioned right above the liquid drop outlet of the first chamber.

16. A droplet generator according to claim 15, wherein: a gap of 0.1-5 mm is formed between the liquid drop output port of the first chamber and the liquid inlet and outlet at one end of the second chamber.

17. A droplet generator according to claim 13, wherein: the liquid drop generating device further comprises a vibration transmission cover, the vibration device is connected with the vibration transmission cover, and the vibration transmission cover is sleeved outside the first chamber.

18. A droplet generator according to claim 13, wherein: the droplet generating device further comprises a pump for providing the driving force required by each liquid flow.

19. A droplet generation apparatus according to claim 18, wherein: the pump can be connected with the first chamber and the second chamber through flexible connecting pieces.

20. A droplet generator according to claim 13, wherein: the first chamber is detachably connected with the second chamber, the first chamber comprises an interface connected with the first cavity, and the second chamber can pass through the interface.

21. A droplet generator according to claim 13, wherein: the second chamber comprises a pipe body with openings at two ends, the openings at the two ends form the liquid inlet and the liquid outlet respectively, the lower part of the pipe body is narrowed close to the openings to form the liquid inlet and the liquid outlet which are narrower than other parts of the pipe body.

22. A droplet generator according to claim 21, wherein: the second room still including set up be in the body on one, two or more be used for connecting the connecting portion of other pipelines, droplet generator still optionally include through the connecting portion with the body one or more feed liquor pipeline of switch-on, second room and one or more feed liquor pipeline be used for letting in respectively the different component parts of second liquid.

23. A droplet generator according to claim 21, wherein: the tube body comprises an outer tube body and an inner tube body which is positioned in the outer tube body and is concentrically arranged with the outer tube body, a liquid inlet and a liquid outlet at the lower end of the inner tube body are positioned above the liquid inlet and the liquid outlet at the lower end of the outer tube body, and the outer tube body and the inner tube body are used for respectively introducing different components of the second liquid.

24. A droplet generator according to claim 21, wherein: the tube body comprises a plurality of at least partially side-by-side tube dividing bodies, and the tube dividing bodies are used for respectively introducing different components of the second liquid.

25. A droplet generator according to claim 13, wherein: a valve is arranged at the liquid drop output opening of the first chamber.

26. A droplet generator according to claim 13, wherein: the apparatus also includes a collector for receiving the generated droplets.

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