CN114377738A - Droplet generation method, system and application thereof - Google Patents

Abstract

The invention relates to a method and a system for generating liquid drops and application thereof, wherein the method comprises the following steps: s1, transferring a second liquid into the droplet-forming tube, the second liquid being immiscible with the first liquid; s2, inserting the liquid drop generating pipe into the first liquid, and enabling the second port of the liquid drop generating pipe to be positioned below the liquid level of the first liquid; s3, controlling the containing cavity to make the volume change periodically, and injecting driving fluid into the fluid path to drive the second liquid to move. The invention provides a brand-new liquid drop generation method, breaks through the limitation that the existing nano-liter level liquid drop generation technology must adopt a micro-pipeline with the diameter less than 0.1mm, and can realize the preparation of uniform liquid drops with small volume by obviously reducing the cost.

Description

Droplet generation method, system and application thereof

Technical Field

The invention belongs to the technical field of liquid drop generation, and particularly relates to a novel liquid drop generation method, a system for the method, and application of the liquid drop generation method and the liquid drop generation system in the fields of clinical diagnosis, gene expression analysis, microorganism detection and the like.

Background

Extracting purified nucleic acid (DNA/RNA template) and Mix from a sample by Polymerase Chain Reaction (PCR) in terms of biomolecule diagnosis; mixing the probe and the primer, and dividing the liquid into uniform liquid drops; each liquid drop contains several or 0 templates, signal amplification detection is carried out after PCR reaction, and absolute quantification of pathogens in a sample is achieved through Poisson distribution calculation, namely digital PCR. The digital PCR detection method has important application in the fields of gene mutation detection and analysis, target drug concomitant detection, cancer marker detection, next generation gene sequencing and the like.

With the development of science and technology, the combination of microfluidics and micro-droplets provides a new direction for micro-reaction and micro-extraction. In these applications, stable and uniform preparation of micro-droplets is important. The traditional droplet preparation technology mostly uses micro-pipes, and utilizes the mutual shearing force formed by oil phase and liquid phase to form water-in-oil micro-droplets, or uses micro-pores formed by physical cutting to divide the droplets.

The patent with publication number CN 112439470A discloses a sample adding needle for preparing micro-droplets and a method for preparing micro-droplets, wherein the sample adding needle adopts a design that 'an upper part is provided with an adaptive part for connecting a liquid supply adapter, a middle part is provided with a liquid storage part for storing a sample, a lower part is provided with a liquid discharge part for generating micro-droplets, an upper end opening of the adaptive part is a liquid supply opening, a lower end opening of the liquid discharge part is a liquid discharge opening, the diameters of the liquid discharge openings are gradually reduced from the liquid supply opening to the liquid discharge opening, the inner diameter of each liquid discharge opening is 25-200 micrometers, the outer diameter of each liquid discharge opening is 200-800 micrometers, a contact angle is not less than 80 degrees when the sample adding needle is contacted with a sample solution and the oily liquid', and then the sample solution in the sample adding needle enters the oily liquid, so that the generation of micro-droplets is realized. Obviously, the inner diameter, thickness, taper, angle and the like of the sample adding pipe are all strictly required, and the processing cost is high.

When the device and the method for preparing uniform single emulsion droplets at high flux are used for preparing droplets, driving phase liquid is continuously introduced into a cavity to form a stable driving phase flow field, then dispersing phase liquid is continuously introduced into an inlet end of a dispersing phase liquid channel, an actuating unit inputs disturbance to the dispersing phase liquid in the dispersing phase liquid channel through a disturbance interface to realize control of jet flow crushing of the dispersing phase liquid, and the dispersing phase liquid is discharged from an outlet end of a capillary structure, wrapped by the driving phase liquid and discharged from a discharge hole to form single emulsion droplets. When the liquid drop is prepared, the dispersed phase liquid and the driving phase liquid are in a jet state, the control of the first two jets is difficult, and the generated liquid drop needs to be formed in the air when being collected, is polluted by the influence of external factors, and is not beneficial to collection and subsequent analysis; further, it is known from the structural characteristics that the droplet generation method is not suitable for droplet preparation in the field of biological detection such as digital PCR droplets or single cell droplets, because the sample size to be detected is usually small and is not enough to form a stable and controllable jet as described in the patent. In addition, the device itself is relatively complex in structure and very costly.

Disclosure of Invention

The invention provides a novel liquid drop generation method in order to overcome the problems of liquid drop preparation of the existing digital PCR liquid drop, single cell liquid drop and the like.

The invention further provides a novel liquid drop generating device and a liquid drop generating system. And further provides the application of the compounds in the fields of clinical diagnosis, gene expression analysis, microorganism detection and the like on the basis.

In order to solve the technical problems, the invention adopts a technical scheme that:

a method of droplet generation employing a droplet generation device and a droplet receiver, a first liquid being disposed within the droplet receiver, the droplet generation device comprising a fluid pathway, a variable volume receiving chamber, and a droplet generation tube having first and second relatively remote ports, wherein the first port communicates with the receiving chamber, the method comprising the steps of:

s1, transferring a second liquid into the droplet-forming tube, the second liquid being immiscible with the first liquid;

s2, inserting the liquid drop generating pipe into the first liquid, and enabling the second port of the liquid drop generating pipe to be positioned below the liquid level of the first liquid;

s3, controlling the containing cavity to make the volume change periodically, and injecting driving fluid into the fluid path to drive the second liquid to move.

According to a further aspect of the droplet generation method of the present invention, in step S3, the droplets are formed within the droplet generation tube and then flow out of the second port and into the droplet receiver. This is very unique, in contrast to prior art droplet generation methods, which always form droplets outside of the microchannel.

According to a further aspect of the droplet generating method of the present invention, in step S3, the droplet generating tube and the droplet receiver may be kept relatively stationary, and the droplet generating tube does not need to be driven to oscillate back and forth as in the prior art.

According to a further aspect of the droplet generation method of the present invention, in step S3, the periodic variation of the holding chamber is represented by a regular variation, which may be a compression-recovery reciprocating variation, or an expansion-recovery reciprocating variation, or a compression-recovery-expansion-recovery reciprocating variation. In one embodiment, the cyclic variation is such that the volume of the holding chamber is reduced from V0 to V1 and then returned from V1 to V0, and so on.

According to a further aspect of the droplet generation method of the invention, the second port has an inner diameter of not less than 0.1 mm. Preferably, the second port has an inner diameter greater than 0.2 mm, particularly preferably between 0.2 mm and 1 mm. More preferably, the second port has an inner diameter of 0.3-0.6 millimeters. This is very unique in contrast to the prior art, where the inner diameter of the outlet of the microchannel is always below 0.1mm in order to obtain micro-droplets. The unique characteristics bring remarkable advantages to the scheme of the invention, firstly, the preparation of uniform liquid drops is less influenced by the processing precision of the micro-pipeline, the uniformity controllability of the liquid drops is obviously improved, and secondly, the processing and manufacturing cost is remarkably reduced. This unique feature is due to the fact that the droplet generation principle of the present invention is distinct from the existing droplet generation principle. Based on the droplet generation principle of the present invention, the inner diameter of the second port cannot be less than 0.1mm, otherwise no droplet can be generated, and more than 0.2 mm is preferable, and basically, when the inner diameter of the second port exceeds 0.2 mm, a uniform small droplet can be stably obtained under a relatively wide range of conditions, especially 0.3 mm to 0.6 mm, where the applicable range of conditions is the broadest.

Preferably, the first port has an inner diameter greater than an inner diameter of the second port.

Preferably, the droplet generating tube comprises a tapered tube portion having both ends respectively forming the first port and the second port, and the taper of the tapered tube portion is 0.05-0.2. Here, the taper refers to a ratio of a difference between an inlet diameter (inner diameter of the first port) of the tapered tube portion and an outlet diameter (inner diameter of the second port) of the tapered tube portion to a length of the tapered tube portion. In the droplet generating method according to the present invention, the taper of the tapered pipe portion has an important influence on generation of droplets, and it is found that stable droplets can be obtained even at a high periodic variation frequency (for example, vibration frequency) by adjusting the taper. Therefore, the frequency range applicable to the device can be expanded through the taper design, and wider requirements can be met.

Preferably, the frequency of the periodic variation is 10Hz-1KHz, preferably 50Hz-600Hz, further preferably 80Hz-600Hz, more preferably 100Hz-600Hz, further preferably 150Hz-500 Hz; particularly preferably 150Hz to 300 Hz.

According to still another embodiment and preferred aspect of the present invention, in step S3, the receiving chamber, the droplet generating tube and the droplet receiver are sequentially disposed from top to bottom, the first port of the droplet generating tube communicates with the bottom of the receiving chamber, and the centerline of the receiving chamber, the axial line of the droplet generating tube, the centerline of the first port and the centerline of the second port are coincident and all extend in the vertical direction.

According to a further embodiment and preferred aspect of the present invention, at least a portion of the wall constituting the accommodating chamber is a movable portion which is moved outwardly or inwardly by an external force to increase or decrease the volume of the accommodating chamber.

Preferably, the movable portion is composed of a metal or nonmetal film.

Further, the movable part is arranged on one or more of the top or the peripheral side of the accommodating cavity. In a preferred embodiment, the movable portion is disposed on a single side wall of the accommodating cavity, and further preferably, the movable portion is disposed on a top side of the accommodating cavity to form a top wall of the accommodating cavity.

In some preferred embodiments, the movable portion is connected to the vibration mechanism through a connection mechanism, so that in step S3, the movable portion is driven by the vibration mechanism to synchronously vibrate back and forth to control the volume of the accommodating cavity to change periodically. Preferably, the direction of the reciprocating vibration is an up-down direction.

In another preferred embodiment, the vibration mechanism is abutted against the movable portion, and the reciprocating vibration of the vibration mechanism is transmitted to the movable portion to generate vibration, thereby controlling the volume of the accommodating chamber to change periodically. Preferably, the direction of the reciprocating vibration is an up-down direction.

The type of vibrating mechanism according to the invention may be, for example, those already used in the prior art, for which the invention is not particularly demanding. However, as a preferred embodiment of the present invention, a vibration frequency of 10Hz to 1KHz may be used when droplet generation is performed; the vibration amplitude that can be used is from 5 microns to 1000 microns. Further preferably, the vibration frequency is 50Hz to 600Hz, further preferably 80Hz to 600Hz, further preferably 100Hz to 600Hz, still further preferably 150Hz to 500Hz, and still further preferably 150Hz to 300 Hz. In some embodiments, the frequency of vibration is 100-300 Hz. The amplitude of the vibration is preferably 5 to 600 micrometers, more preferably 5 to 300 micrometers, further preferably 5 to 100 micrometers, and further preferably 5 to 60 micrometers. In some embodiments, the amplitude of the vibrations is from 5 microns to 50 microns, and in other embodiments, the amplitude of the vibrations is from 20 microns to 60 microns.

According to a further preferable mode of the present invention, when the taper of the tapered tube portion is 0.05 to 0.1, the vibration frequency is set to 100Hz to 600Hz, and the amplitude of the vibration is set to 10 micrometers to 300 micrometers. When the taper of the tapered tube part is 0.1-0.2, the frequency of the vibration is set to be 100-300HZ, and the amplitude of the vibration is 10-600 microns.

Preferably, in step S3, the fluid is a liquid. Further, the injection speed is 2-200. mu.l/min, preferably 10-50. mu.l/min.

Preferably, the accommodating cavity is an annular cavity with an inner diameter of 4-6 mm. Further, it is preferable that the inner peripheral side wall of the accommodation chamber is provided to extend in the vertical direction. It is also preferable that the inner wall surface of the accommodating chamber is a smooth surface.

Further, according to the present invention, step S1 may be performed before step S2, or step S1 may be performed after step S2, and the order therebetween has no influence on the generation effect of the droplets.

According to a specific embodiment and preferred aspects of the present invention, in step S1, the second liquid is drawn in from the second port of the droplet-generating tube, and thereafter, a part of the first liquid is drawn in, or the first liquid is not drawn in.

In some embodiments, a second liquid is drawn in from the second port of the droplet-generating tube and a portion of the first liquid is drawn in thereafter, such that the liquid within the droplet-generating tube is divided into a driving-fluid segment, a second-liquid segment, and a first-liquid segment in order from top to bottom prior to beginning step S3. This has the advantage that the uniformity of the droplets produced throughout can be improved.

In some embodiments, the second liquid is drawn into the second port of the droplet-generating tube and then the first liquid is not drawn into the second port, such that the liquid in the droplet-generating tube is divided into a driving-fluid segment and a second-liquid segment from top to bottom in sequence before starting step S3.

According to still another embodiment and preferred aspect of the present invention, the droplet generating method further comprises a step of cleaning and/or discharging bubbles from the holding chamber and the droplet generating tube after completion of one droplet generation or before starting of the next droplet generation.

Preferably, two plunger pumps with different volume sizes are respectively used for controlling the driving fluid, and the switching control is performed by combining a three-way valve, wherein the plunger pump with the larger volume is used for the step of cleaning and/or bubble discharging, and the plunger pump with the smaller volume is used for the step of forming liquid drops.

According to the invention, the first liquid is typically the continuous phase and the second liquid is typically the dispersed phase; and/or the first liquid is typically an oil phase and the second liquid is typically an aqueous phase.

According to yet another specific implementation and preferred aspect of the invention, a surfactant is added to the first liquid; the second liquid is an aqueous phase containing the biological or chemical substance to be detected.

Preferably, the droplets are digital PCR droplets or single cell droplets. The diameter of the droplets is 50 to 250 micrometers, preferably 200 micrometers or less, more preferably 150 micrometers or less, further preferably 120 micrometers or less, and further preferably 110 micrometers or less.

The invention adopts another technical scheme that: a liquid drop generating system comprises a liquid drop generating device and a liquid drop receiver, wherein the liquid drop receiver is used for containing first liquid and liquid drops, the liquid drop generating device comprises a containing cavity with a variable volume, a control mechanism used for controlling the volume of the containing cavity to be changed periodically, and a liquid drop generating pipe with a first port and a second port which are far away from each other, the first port of the liquid drop generating pipe is communicated with the containing cavity, the liquid drop generating device further comprises a driving fluid mechanism used for introducing driving fluid into the containing cavity, and the inner diameter of the second port of the liquid drop generating pipe is more than 0.1 millimeter.

As set forth above, the inner diameter of the second port is preferably greater than 0.2 mm, particularly preferably 0.2 mm to 1 mm. More preferably, the second port has an inner diameter of 0.3-0.6 millimeters. Also preferably, the inner diameter of the first port is greater than the inner diameter of the second port. The liquid drop generating pipe comprises a conical pipe part, the first port and the second port are formed at two ends of the conical pipe part respectively, and the taper of the conical pipe part is 0.05-0.2.

Further, the volume of the droplet generating tube may be 10 to 200. mu.l. Experiments show that the volume of the droplet generation tube has less influence on the droplet generation effect, which is one of the advantages of the droplet generation method of the invention.

According to a specific implementation and preferred aspect of the invention, the periodic variation is a compression-recovery reciprocation, or an expansion-recovery reciprocation, or a compression-recovery-expansion-recovery reciprocation.

According to a specific implementation and preferred aspect of the invention, the driving fluid mechanism comprises a pump and a fluid passage, the droplet generating device comprises a base body, the base body provides a cylindrical hole and a fluid passage, and provides a connecting part for connecting the liquid generating tube, the number of the cylindrical hole, the number of the fluid passage and the number of the connecting part are one or more, the cylindrical hole is a cylinder with an opening at the upper part and the lower part, a membrane is covered on each cylindrical hole, and the cylindrical hole and the membrane jointly form a containing cavity.

Preferably, the accommodating cavity, the liquid drop generating pipe and the liquid drop receiver are sequentially arranged from top to bottom, the first port of the liquid drop generating pipe is communicated with the lower opening of the accommodating cavity, and the central line of the accommodating cavity, the axial line of the liquid drop generating pipe, the central line of the first port and the central line of the second port are overlapped and extend along the vertical direction.

Preferably, each diaphragm comprises a main body part and a movable part, the main body part is fixedly connected with the base body, the movable part is positioned right above the cylindrical hole, and the movable part is connected to the control mechanism through a connecting part.

Further, the diaphragm may be a metal (e.g., stainless steel) or non-metal diaphragm; the membrane may have a thickness of 5 microns to 2 mm.

Preferably, a sealing structure is provided between the membrane and the base body to maintain a good seal of the receiving chamber at all times.

Preferably, the control mechanism is a vibration mechanism, and the vibration mechanism includes one or more of a galvanometer motor, piezoelectric ceramics, and a voice coil motor, and is not particularly limited. According to a particularly preferred aspect of the present invention, the vibrating mechanism preferably comprises a piezoelectric ceramic.

Preferably, the vibration mechanism provides a vibration direction in an up-and-down direction, a vibration frequency provided including at least 50-600Hz, which may be particularly selected within this range in use, and a vibration amplitude provided including at least 5-300 microns.

Preferably, the droplet generating tube is detachably connected to the connecting portion. In this way, the droplet generation tube portion can be independent of the entire system, and can be removed and replaced when droplet generation of a sample solution is completed. In some embodiments, the droplet generation tube may be a commercially available nozzle with a corresponding specification. Of course, it is also possible to integrate the droplet generation tube with the connection portion without affecting the droplet generation effect.

Preferably, on the same base body, the number of the cylindrical holes, the number of the fluid passages and the number of the connecting parts are respectively 2-20, and preferably 4-10.

Preferably, the plurality of cylindrical holes, the plurality of fluid passages and the plurality of connecting portions are provided, two opposite sides of the base are higher than a middle portion between the two opposite sides, the plurality of cylindrical holes are independently distributed in the middle portion of the base and are arranged in two rows, and each fluid passage includes a vertical passage formed on the two opposite sides of the base and a horizontal passage correspondingly communicating the vertical passage with the cylindrical holes. By adopting the structural design, the device is compact on the whole and convenient to operate and control.

According to a specific implementation and preferred aspect of the invention, a drainage portion is formed between the port of the horizontal passage and the inner peripheral side wall of the accommodating cavity, so that liquid from the horizontal passage enters the accommodating cavity in a tangential direction to the circumferential direction of the accommodating cavity.

According to a specific embodiment and preferred aspect of the present invention, an end portion of the fluid passage communicates with the accommodating chamber of the base body, and when the fluid is driven from the fluid passage into the accommodating chamber, the fluid forms a vortex in the accommodating chamber and the droplet generating tube, the vortex rotating in a circumferential direction of the accommodating chamber and the droplet generating tube.

According to still another embodiment and preferred aspect of the present invention, an end portion of the fluid passage communicates with the housing chamber of the base, and a direction in which the fluid is discharged from the fluid passage is offset from an axial center line of the housing chamber.

Above, through structural design such as fluid passage and holding chamber, can make on the whole can be so that can conveniently discharge the bubble in the system cavity before carrying out the liquid droplet and generate, avoid the bubble to have the adverse effect to the liquid droplet generation.

According to the present invention, the droplet receiver is a device having a receiving chamber, and may be in a closed or non-closed form, without particular limitation.

The invention adopts another technical scheme that: a droplet generating apparatus is configured in the same manner as the droplet generating apparatus in the droplet generating system described above, except that it does not include a droplet receiver.

The other technical scheme of the invention is as follows: the application of the liquid drop generating method, the liquid drop generating system and the liquid drop generating device in clinical diagnosis, gene expression analysis and microorganism detection.

In the present invention, the oil phase is not particularly limited and may be any suitable oil phase reported in the prior art. Preferably, the oil phase comprises an oil and a surfactant. Specifically, the oil may be, for example, one or a combination of more of fluorocarbon oil, silicone oil, mineral oil, hydrocarbon oil, and vegetable oil. The surfactant typically has a Hydrophilic Lipophilic Balance (HLB) of 3 to 6. The content of the surfactant in the oil phase is generally 0.1-20 wt%, preferably 0.1-10 wt%.

Surfactants suitable for generating water-in-oil droplets have been reported in the prior art in large numbers and are suitable for use in the oil phase of the present invention.

As a specific and preferred aspect of the present invention, when the oil is a fluorocarbon oil, the surfactant is a fluorine-containing surfactant, specifically, for example, perfluorooctanol, perfluorodecanol, perfluorotetradecanoic acid, perfluoropolyether carboxylic acid and derivatives thereof, and the like.

As still another specific and preferred aspect of the present invention, when the oil is a silicone oil, the surfactant may be a nonionic surfactant such as polyethylene glycol octyl phenyl ether (triton x-100), a silicone chain nonionic surfactant such as ABIL EM90, an anionic surfactant such as SDS, or the like.

As a further specific and preferred aspect of the present invention, when the oil is a hydrocarbon oil, the surfactant may be a nonionic surfactant such as tween 20, tween 80, a silicone chain nonionic surfactant such as ABIL EM90, an anionic surfactant such as SDS, a phospholipid or the like.

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

the invention provides a brand-new liquid drop generation method, breaks through the limitation that the existing nano-liter level liquid drop generation technology must adopt a micro-pipeline with the diameter less than 0.1mm, and can realize the preparation of uniform liquid drops with small volume by obviously reducing the cost.

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 a schematic structural view of a liquid droplet generating apparatus of embodiment 1;

FIG. 2 is a schematic view of a portion of the structure of FIG. 1;

FIG. 3 is a schematic view of a portion of the structure of FIG. 2;

FIG. 4 is a schematic cross-sectional view of FIG. 3;

FIG. 5 is a schematic cross-sectional view of a single droplet generation unit in the droplet generation apparatus of example 1;

FIG. 6 is a schematic sectional view taken along line A-A in FIG. 5;

FIG. 7 is a schematic cross-sectional structural view of a liquid droplet generating system of example 1;

FIG. 8 is a schematic sectional view of a single liquid droplet-generating unit in example 1 filled with a driving oil;

FIG. 9 is a schematic cross-sectional view of the formation of a drive fluid segment and a second liquid segment in a single droplet-generating unit of example 1;

FIG. 10 is a schematic cross-sectional structural view of a liquid droplet generating system of example 2;

FIGS. 11 to 16 are microscope images of the droplets prepared in examples 3 to 7, respectively;

FIG. 17 is a schematic diagram showing the velocity field distribution near the exit of a droplet-generating tube;

in the drawings: 1. a droplet generating device; 10. a substrate; 10a, a middle part; 10b, a left convex edge part; 10c, right raised edge portion; 100. a cylindrical hole; 101. a connecting portion; t, a channel; 11. a droplet generation unit; 110. an accommodating chamber; 111. a control mechanism; 112. a droplet generating tube; a1, a first port; a2, a second port; c1, connecting pipe part; c2, a tapered tube portion; 113. a drive fluid mechanism; d1, pump; d2, fluid pathway; d21, vertical access; d22, horizontal path; d3, a drainage part; 114. a membrane; b1, a main body part; b2, a movable part; 115. a connecting member; y1, a first liquid; y2, a second liquid; y3, fluid; 116. a seal ring; 2. a droplet receiver.

Detailed Description

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 stated 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 specific cases to those skilled in the art.

In the prior art, it is generally considered advantageous that the microchannels for generating microdroplets are small, and therefore, the inner diameter of microchannels actually used is generally below 0.1 mm. Meanwhile, the generation technology of micro-droplets in the prior art mainly comprises a co-current focusing mode and a micro-pipeline vibration mode, for the co-current focusing mode, a dispersed phase liquid is ejected to a continuous phase in a vibration mode and is wrapped by the continuous phase, and then the dispersed phase liquid is ejected from an ejection hole together to form droplets, and for the micro-pipeline vibration mode, the micro-pipeline (sample adding pipe) needs to vibrate back and forth to generate the droplets.

The inventors of the present invention have found in a great deal of experimental studies that the preparation of uniform droplets is achieved only by combining the dispersion phase vibration and the micro-channel of appropriate inner diameter. Based on the discovery, the inventor further researches the mechanism of the liquid drop generating device, carries out simulation structure analysis on key influencing factors influencing the liquid drop generation, and verifies the key influencing factors through further experiments. Research shows that the liquid drop generation of the invention has a brand new generation principle which is obviously different from any liquid drop generation mode in the prior art, and compared with the existing liquid drop generation mode, the liquid drop generation mode can really realize the generation of nano-liter volume micro-drops by a structure with non-microfluidic scale (the geometric scale of all cavities and consumables related to the generation process disclosed by the invention is more than 0.1 millimeter, and generally 0.1 millimeter is the critical size for distinguishing microfluidic). The structure scale actually used by the droplet generation technology in the fields of digital PCR, single cell sorting and the like in the prior art is smaller than or close to the diameter of 0.1mm of a nanoliter droplet. The droplet generation technology can generate the nanometer droplets by using the structure which is far larger than the nanometer droplet size, and is a core technical breakthrough for realizing the use cost of the micro-droplet type digital PCR. Based on the droplet generation technology, even a common pipette tip can be directly adopted as a key generation consumable. This droplet generation technique, which is called non-vibrational ejection (non-mechanical ejection), has no mechanical motion other than the micro motion of the dispersed phase. The invention discloses a technology for generating liquid drops by destabilizing a dispersed phase due to a velocity gradient in a liquid drop generating pipeline, which realizes the control of nano-liter precision.

The droplet generation principle of the present invention is described as follows:

the driving mechanism (e.g., a vibrating mechanism) pushes and pulls the movable part by directly connecting with the movable part (e.g., an elastic membrane) or touches the movable part by contacting with the movable part, so that the movable part generates periodic vibration to drive the liquid (second liquid/dispersed phase/water phase) in the accommodating cavity to generate periodic motion, and meanwhile, the driving fluid (driving oil) is continuously injected into the cavity, and then the periodic motion including forward pushing ejection and back pumping motion is generated at an oil-water interface (interface between the first liquid and the second liquid) of the outlet (second port) of the droplet generation tube. In the forward propelling ejection stage, according to the characteristics of the tubular fluid, it can be known that the flow velocity in the middle of the tube is greater than the flow velocity near the wall surface (as shown in fig. 17, where the velocity field distribution of the outlet of the droplet generation tube is shown, the brightness represents the velocity, and the velocity at the center of the outlet is the maximum, which stretches the oil-water interface), so that the moving velocity of the middle interface is greater than the moving velocity of the interface near the wall surface, so that the oil-water interface forms a tapered interface, which is continuously stretched. This is also the key reason that the outlet of the droplet generation tube is much larger than the droplet diameter (-0.1 mm). Subsequent withdrawal movement pulls the tapered interface back through a periodic movement. It should also be noted that the exact location of droplet generation within the droplet generation conduit (in some specific experiments, droplets are generated at a distance of about 0.5mm from the outlet) was found to be different from other generation techniques in which droplet formation is outside the conduit outlet. This also represents the uniqueness of the present technology.

Compared with a droplet generation method adopting continuous high-frequency vibration of a micro-pipeline in oily liquid, the vibration-free ejection technology based on the invention has obvious advantages, on one hand, the depth of the micro-pipeline extending into the oily liquid does not need strict requirements, and the generated droplets are not damaged, so that the quality and the generation operation of the droplets in the whole generation process are more controllable; on the other hand, in the prior art, the requirements on the inner diameter of the micro-pipeline are all within 0.1mm, the larger the inner diameter is, the higher the high-frequency swing frequency is required to be applied, the control requirement is high, the stability is poor, and meanwhile, the requirement on the consistency is also higher during the manufacturing of the micro-pipeline, the vibration is applied to the aqueous phase liquid, the generation of liquid drops is realized, the requirement on the inner diameter of a liquid inlet and a liquid outlet of the micro-pipeline is reduced (can be more than 0.1mm, preferably more than 0.3 mm), the uniformity of the liquid drops can be ensured, the control is easier, the processing difficulty of the sampling tube is obviously reduced, and the processing cost is reduced. Compared with a co-flow focusing mode, the invention has the advantages that the structure of the system can be greatly simplified, the structure is simpler, the operation is more convenient and the cost is obviously reduced on the premise of realizing the uniformity and the stability of the liquid drop generation.

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.

Example 1

As shown in fig. 1 to 9, the droplet generation system according to the present embodiment includes a droplet generation device 1 and a droplet receiver 2.

The liquid drop generating device 1 comprises a substrate 10 and a plurality of liquid drop generating units 11 arranged on the substrate 10, wherein the plurality of liquid drop generating units 11 are arranged in parallel, and single-path or multi-path simultaneous liquid drop generation can also be realized.

Referring to fig. 2, the substrate 10 is an aluminum block and is in a strip shape, and a middle portion 10a, a left protruding edge portion 10b, and a right protruding edge portion 10c are formed by recessing from the middle of the strip shape downward from the surface, wherein the plurality of droplet generation units 11 are distributed side by side and at regular intervals along the length direction of the substrate 10.

Referring to fig. 3 and 4, eight cylindrical holes 100 extending up and down are formed in the middle portion 10a of the base 10, a connecting portion 101 is correspondingly formed below each cylindrical hole 100, the eight cylindrical holes 10 are divided into two rows, four cylindrical holes 10 in each row are uniformly distributed at intervals, and the left and right rows of cylindrical holes 10 are aligned left and right.

As shown in fig. 5, each of the droplet generating units 11 includes a variable volume accommodating chamber 110, a control mechanism 111 for controlling the volume of the accommodating chamber 110 to be periodically changed, a droplet generating tube 112 having a first port a1 and a second port a2 which are relatively far away, and a driving fluid mechanism 113 for introducing a driving fluid into the accommodating chamber 110.

In this example, a diaphragm 114 is placed over the cylindrical hole 100, and the cylindrical hole 100 and the corresponding diaphragm 114 together form the receiving cavity 110. The control mechanism 111 is a vibration mechanism which is installed above the cylindrical hole 100 and is connected with the diaphragm 114 through a connection member 115, thereby controlling the movement of the diaphragm 114 to implement the volume change of the accommodation chamber 110. The droplet generating tube 112 is vertically disposed, and has an upper end portion communicating with the connection portion 101 and a lower end portion forming a droplet outlet. The driving fluid mechanism 113 is provided at the left or right convex edge portion 10b or 10c to which the cylindrical hole 100 is close.

The center line of the accommodation chamber 110, the axis line of the droplet generation tube 112, the center line of the first port a1, and the center line of the second port a2 coincide and extend in the vertical direction. The receiving chamber 110 is an annular chamber having an inner diameter of about 5mm, and an inner circumferential side wall extending in a vertical direction. The diaphragm 114 (made of stainless steel) forming the top of the accommodating chamber 110 includes a main body portion b1 and a movable portion b2, wherein the main body portion b1 is fixedly connected to the base 10, the movable portion b2 is located right above the cylindrical hole 100, and the movable portion b2 is connected to the control mechanism 111 through a connecting member 115. The control mechanism 111 specifically includes a piezoelectric ceramic, which provides up-and-down reciprocating vibration, and when the piezoelectric ceramic reciprocates, the movable portion b2 is driven to move inwards or outwards relative to the accommodating cavity 110, so as to periodically change the volume of the accommodating cavity 110, and accordingly, the liquid in the accommodating cavity 110 is disturbed due to the periodic volume change. Further, the periodic variation may be a compression-recovery reciprocation variation, or an expansion-recovery reciprocation variation, or a compression-recovery-expansion-recovery reciprocation variation. In this example, at least the periodic variation that may be provided includes a reciprocating variation of compression-recovery. A further piezoceramic 40VS12 (with internal threads interfacing with connector 115).

The droplet generating tube 112 includes a connecting tube portion c1 and a tapered tube portion c 2. The conical tube portion c2 has a first port a1 and a second port a 2. The inner diameter of the tapered tube portion c2 gradually decreases from the first port a1 toward the second port a2, and the change in the inner diameter exhibits a taper which has an important effect on the generation effect of liquid droplets. Assuming that the first port a1 has an inner diameter R1, the second port a has an inner diameter R2, the distance between the first port a1 and the second port a2 (i.e., the length c1 of the tapered tube portion) is L, and the taper is R1-R2/L. In this example, the taper of the tapered tube portion c2 is 0.12, and the inner diameter R2 of the second port a2 is 0.5. + -. 0.1 mm. The volume of the droplet generating tube 112 was about 10. mu.l.

The connecting tube portion c1 intersects the tapered tube portion c2 at the first port a1 for detachable fitting over the connecting portion 101, and its taper is not particularly required, but is preferably greater than that of the tapered tube portion c 2. Wherein a flow passage t communicating with the receiving chamber 110 is formed at the middle of the connecting portion 101. After the droplet generating tube 112 is attached to the connecting portion 101, the housing chamber 110 communicates with the droplet generating tube 112 through the flow path t.

As shown in fig. 6, the driving fluid mechanism 113 includes a pump d1 and a fluid passage d2, wherein the fluid passage d2 includes a vertical passage d21 formed on the left raised edge portion 10b of the base 10 and a horizontal passage d22 that correspondingly communicates the vertical passage d21 with the cylindrical hole 100. To facilitate the discharge of air bubbles in the system cavity, a drain d3 is formed between the port of the horizontal passage d22 and the inner peripheral side wall of the accommodating chamber 110 so that the liquid from the horizontal passage d2 enters the accommodating chamber 110 tangentially to the circumferential direction of the accommodating chamber 110, and thus the fluid forms a vortex in the accommodating chamber 110 and the droplet generating tube 112 rotating in the circumferential direction of the accommodating chamber 110 and the droplet generating tube 112, whereby air bubbles can be easily discharged. In other embodiments, the drainage portion is not necessarily provided, and the bubble discharge effect can be preferably achieved as long as the direction in which the fluid is discharged from the fluid passage d22 is deviated from the axial line of the accommodating chamber.

In this example, two plunger pumps having different volume sizes are used to control the driving fluid, and a three-way valve is used to perform switching control, wherein the plunger pump having a larger volume is used in the step of washing and/or discharging bubbles, and the plunger pump having a smaller volume is used in the step of forming droplets.

In this example, a sealing ring 116 is also provided between the diaphragm 114 and the base body 10 to improve the sealing of the receiving chamber.

As shown in fig. 7, the droplet receiver 2 is located below the second port a2 and is configured to receive the first liquid y1 and the droplet.

In the field of digital PCR, the first liquid y1 is typically a formulated oil, such as mineral oil, preferably with the addition of a surfactant. The second liquid y2 is typically an aqueous phase of the biological or chemical substance to be detected.

As shown in fig. 8, fluid y3 (drive oil) fills the inner cavities of fluid passage d2, accommodating chamber 110, and droplet generation tube 112. Further, the same mineral oil may be used for the fluid y3 and the first liquid y 1.

As shown in fig. 9, in general, when droplet generation is performed, the liquid in the droplet generation tube 112 is divided into a driving fluid segment and a second liquid segment from the top in this order. Or, the liquid in the droplet generation tube 112 is sequentially divided into a driving fluid segment, a second liquid segment and a first liquid segment from top to bottom; thereafter, the drive mechanism and the fluid drive mechanism are turned on to start droplet generation.

Example 2

As shown in fig. 10, the present embodiment provides a droplet generating apparatus, which is substantially the same as embodiment 1, except that the length from the first port a1 to the second port a2 of the droplet generating tube is 2L, and accordingly, the taper of the tapered tube portion c2 of the droplet generating tube 112 corresponds to 0.06, that is, the taper of the tapered tube portion of the droplet generating tube 112 in this embodiment is half of the taper of embodiment 1.

Example 3

The droplet generation method of the present embodiment adopts the droplet generation system of the present embodiment, and the implementation process is as follows:

s1, filling a fluid (driving oil) in the fluid passage, the accommodating cavity and the inner cavity of the liquid drop generating pipe by using a pump (plunger pump) with a larger volume, sucking a water phase (second liquid) to be detected into the liquid drop generating pipe through a second port, and sucking a section of formula oil (first liquid), wherein the liquid in the liquid generating pipe comprises a three-section structure, namely a driving oil section, a water phase section and a formula oil section from top to bottom;

s2, inserting the liquid drop generating pipe into the formula oil to enable the second port of the liquid drop generating pipe to be located about 2mm below the liquid level of the formula oil;

s3, under the vibration drive of the vibration mechanism, the movable part of the membrane is directly pushed and pulled to synchronously vibrate up and down in a reciprocating mode, the containing cavity is controlled to enable the volume of the containing cavity to change periodically, and drive oil is injected into the fluid passage to drive the water phase to move, liquid drops are generated in the conical pipe part of the liquid drop generating pipe at the moment, then the formed liquid drops flow out through the second port of the liquid drop generating pipe and enter the liquid drop receiver, the liquid drop generating pipe does not need to be driven to move in the process, and the liquid drop generating pipe and the liquid drop receiver are kept static relatively.

In this example, according to the above steps, water is used as the second liquid, and droplets prepared under the conditions of the injection speed of the driving oil being 36.5 microliters/minute, the vibration frequency of the piezoelectric ceramic being 150Hz, the vibration amplitude being 50 micrometers, and the input voltage being 2.5v are shown in fig. 11 and 12, wherein it can be seen that droplets with very uniform size are successfully prepared, and the diameter of the droplets is between 104 micrometers and 106 micrometers.

Example 4

The droplet generation method of the present example is basically the same as example 3 except that: the injection speed of the drive oil is changed.

Under the conditions that the injection speed of the driving oil is 19.5 microliter/minute, the vibration frequency of the piezoelectric ceramic is 150Hz, the vibration amplitude is 50 micrometers and the input voltage is 2.5v, the diameter of the formed liquid drop is shown in figure 13a, and the diameter of the generated liquid drop is about 85 +/-1 micrometers.

Under the conditions that the injection speed of the driving oil is 15.0 microliter/minute, the vibration frequency of the piezoelectric ceramic is 150Hz, the vibration amplitude is 50 micrometers and the input voltage is 2.5v, the diameter of the formed liquid drop is shown in figure 13b, and the diameter of the generated liquid drop is about 78 +/-1 micrometers.

Example 5

The droplet generation method of the present example is basically the same as example 3 except that: the vibration frequency is changed.

Under the conditions that the injection speed of the driving oil is 48.7 microliters/minute, the vibration frequency of the piezoelectric ceramic is 200Hz, the vibration amplitude is 50 micrometers, and the input voltage is 2.5v, the diameter of the formed liquid drop is shown in figure 14a, and the diameter of the generated liquid drop is 104.00-107.00 micrometers.

Under the conditions that the injection speed of the driving oil is 60.8 microliters/minute, the vibration frequency of the piezoelectric ceramic is 250Hz, the vibration amplitude is 50 micrometers, and the input voltage is 2.5v, the diameter of the formed liquid drop is shown in figure 14b, and the diameter of the generated liquid drop is 103.00-108.50 micrometers.

Under the conditions that the injection speed of the driving oil is 73.0 microliter/minute, the vibration frequency of the piezoelectric ceramic is 300Hz, the vibration amplitude is 50 micrometers and the input voltage is 2.5v, the diameter of the formed liquid drop is shown in figures 14c and 14d, and the diameter of the generated liquid drop is 93.00-106.00 micrometers.

It can be seen from this embodiment that the droplet generation method of the present invention can obtain uniform droplets at different frequencies.

Example 6

In this example, experiments were carried out at different vibration frequencies using the droplet generating apparatus according to example 2, that is, using a droplet generating tube having a smaller taper, and it was found that uniform droplets were obtained even when the vibration frequency of the piezoelectric ceramic was 600 Hz. Comparable to example 3, which produced droplets at 150 Hz.

Example 7

The droplet generation method of the present example is basically the same as example 3 except that: droplet generation was performed using RCR reagent as the second liquid instead of water. RCR reagent 20ul system was used: bole supermix 10ul + Bole demo kit dna 1ul + fam probe 1ul + hex probe 1ul + water 7 ul.

The prepared droplets are shown in fig. 15 and 16, and it can be seen that droplets with very uniform size are successfully prepared, and the diameters of the droplets are all about 104-107 micrometers.

In the above embodiments, the droplet generation tube can be disassembled and replaced after use to prevent cross contamination of different samples, and the above droplet generation scheme can also be applied in the following fields.

Quantification: 1) and analyzing the transgenic components. And (3) detecting microorganisms: 1) detecting microorganisms in the water sample; 2) and detecting pathogenic microorganisms.

As can be seen from the above examples, the present invention has a number of technical advantages in the preparation of droplets, including but not limited to:

1. the uniform and micro liquid drops can be formed through a simple structure, the technical advantages of repeatable and continuous preparation and the like are achieved, the method can be applied to occasions such as clinical diagnosis, gene expression analysis, microorganism detection and the like, and the practicability is good;

2. the liquid drops are generated in the liquid drop generating pipe, under the condition that the device is set, the generating effect of the liquid drops is mainly influenced by the vibration frequency, and the device is insensitive to the tiny change of the inner diameter of the liquid drop generating pipe, the position of the liquid drop generating pipe inserted below an oil phase interface, the formula composition of the oil phase and the like, so the consistency and the controllability of the liquid drop generation are obviously improved;

3. the device is internally provided with a bubble discharging structure and a bubble discharging function, so that bubbles can be effectively discharged while cleaning, and the interference on the generation of liquid drops due to the existence of bubbles is avoided;

4. the requirement for controlling the inner diameter of the liquid drop generating tube is obviously reduced, and the cost can be obviously reduced correspondingly, so that the liquid drop generating tube can be used as a consumable material to prevent mutual pollution among generated samples, and a disposable liquid drop generating tube can be adopted;

5. the prepared liquid drops are directly stored in a liquid drop receiver without transfer, and the prepared liquid drops can be directly subjected to PCR amplification and analysis, so that the integration of liquid drop generation and analysis is realized;

6. the application can divide a sample into micro droplets with uniform sizes for detection, and has the advantages of high specificity, high sensitivity, high precision and the like of a detection result; meanwhile, the detection of a single micro-droplet is more beneficial to the analysis and research of a sample on a microscopic level;

7. the liquid drop generating system and method of the present invention have wide application fields, and the applicable fields include but are not limited to the following aspects:

and (3) clinical diagnosis: 1) noninvasive prenatal diagnosis: detection of fetal genetic disease by maternal episomal DNA fragments; 2) detecting a cancer marker; 3) detecting the virus; 4) analyzing copy number variation; 5) and detecting mutation.

Gene expression analysis (mainly analysis of genetic gene differences between cells): 1) analyzing gene expression; 2) and analyzing the single cell gene expression.

Next generation sequencing aspect: 1) verifying a sequencing result; 2) and sequencing library quality control.

Transgenic component quantification: and (4) analyzing the components of the transgenes.

And (3) detecting microorganisms: 1) detecting microorganisms in the water sample; 2) and detecting pathogenic microorganisms.

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 (39)

1. A method of droplet generation using a droplet generation device and a droplet receiver in which a first liquid is disposed, the droplet generation device comprising a fluid pathway, a variable volume receiving chamber, and a droplet generation tube having first and second relatively remote ports, wherein the first port communicates with the receiving chamber, the method comprising the steps of:

s1, transferring a second liquid into the droplet-forming tube, the second liquid being immiscible with the first liquid;

s2, inserting the liquid drop generating pipe into the first liquid, and enabling the second port of the liquid drop generating pipe to be positioned below the liquid level of the first liquid;

s3, controlling the containing cavity to make the volume change periodically, and injecting driving fluid into the fluid path to drive the second liquid to move.

2. A liquid droplet generation method according to claim 1, characterized in that: in step S3, the droplet is formed within the droplet generation tube and then flows out of the second port and into the droplet receiver.

3. A liquid droplet generation method according to claim 1, characterized in that: in step S3, the droplet generation tube and the droplet receiver are held stationary relative to each other.

4. A liquid droplet generation method according to claim 1, characterized in that: the periodic variation is a compression-recovery reciprocation variation, or an expansion-recovery reciprocation variation, or a compression-recovery-expansion-recovery reciprocation variation.

5. A liquid droplet generation method according to claim 1, characterized in that: in step S3, the accommodating chamber, the droplet generating tube, and the droplet receiver are sequentially disposed from top to bottom, the first port of the droplet generating tube is communicated with the bottom of the accommodating chamber, and the center line of the accommodating chamber, the axial line of the droplet generating tube, the center line of the first port, and the center line of the second port are overlapped and extend in the vertical direction.

6. A liquid droplet generation method according to claim 1, characterized in that: the inner diameter of the second port is not less than 0.1mm, preferably more than 0.2 mm, more preferably 0.2-1 mm, still more preferably 0.3-1 mm, and particularly preferably 0.3-0.6 mm; and/or the inner diameter of the first port is larger than the inner diameter of the second port; and/or the liquid drop generating pipe comprises a conical pipe part, the first port and the second port are respectively formed at two ends of the conical pipe part, and the taper of the conical pipe part is 0.05-0.2; the frequency of the periodic variation is 10Hz-1KHz, preferably 50Hz-600Hz, further preferably 80Hz-600Hz, more preferably 100Hz-600Hz, further preferably 150Hz-500 Hz; particularly preferably 150Hz to 300 Hz.

7. A droplet generation method according to any of claims 1 to 6, wherein: at least a part of the wall forming the accommodating cavity is a movable part which moves outwards or inwards under the action of external force, so that the volume of the accommodating cavity is increased or reduced.

8. The method of generating droplets of claim 7, wherein: the movable part is composed of a metal or nonmetal film; and/or the movable part is arranged in one or more of the top or the peripheral side wall of the accommodating cavity.

9. The method of generating droplets of claim 7, wherein: the movable part is connected with the vibration mechanism through the connecting mechanism, so that in step S3, the movable part is driven by the vibration mechanism to synchronously vibrate in a reciprocating manner to control the volume of the accommodating cavity to change periodically; or the vibration mechanism is abutted against the movable part, and the reciprocating vibration of the vibration mechanism is transmitted to the movable part to generate vibration so as to control the volume of the accommodating cavity to change periodically.

10. A liquid droplet generation method according to claim 9, characterized in that: the reciprocating vibration direction is the up-down direction.

11. A liquid droplet generation method according to claim 10, characterized in that: the amplitude of the vibration is 5 to 1000 micrometers, preferably 5 to 600 micrometers, more preferably 5 to 300 micrometers, further preferably 5 to 100 micrometers, and further more preferably 5 to 60 micrometers.

12. A liquid droplet generation method according to claim 9, characterized in that: when the taper of the conical tube part is 0.05-0.1, setting the vibration frequency to be 100Hz-600Hz, and setting the vibration amplitude to be 10-300 micrometers; when the taper of the tapered tube part is 0.1-0.2, the frequency of the vibration is set to be 100-300HZ, and the amplitude of the vibration is 10-600 microns.

13. A liquid droplet generation method according to claim 1, characterized in that: in step S3, the fluid is a liquid, and the injection speed is 2-200 μ l/min, preferably 10-50 μ l/min.

14. A liquid droplet generation method according to claim 1, characterized in that: the accommodating cavity is an annular cavity, and the inner diameter of the accommodating cavity is 4-6 mm; and/or the inner peripheral side wall of the accommodating cavity extends along the vertical direction.

15. A liquid droplet generation method according to claim 1, characterized in that: the step S1 is performed before the step S2, or the step S1 is performed after the step S2.

16. A liquid droplet generation method according to claim 1, characterized in that: in step S1, the second liquid is sucked in from the second port of the droplet generation tube, and then a part of the first liquid is sucked in, or the first liquid is not sucked in.

17. A liquid droplet generation method according to claim 1 or 16, characterized in that: before starting step S3, dividing the liquid in the droplet generation tube into a driving liquid segment and a second liquid segment from top to bottom in this order; or the liquid in the liquid drop generating pipe is sequentially divided into a driving fluid section, a second liquid section and a first liquid section from top to bottom.

18. A liquid droplet generation method according to claim 1, characterized in that: the method for generating the liquid drops further comprises the step of cleaning and/or discharging bubbles from the accommodating cavity and the liquid drop generating pipe after the generation of one liquid drop is finished or before the generation of the next liquid drop is started.

19. A liquid droplet generation method according to claim 18, wherein: two plunger pumps with different volume sizes are respectively adopted to control the driving fluid, and a three-way valve is combined to carry out switching control, wherein the plunger pump with larger volume is used for the step of cleaning and/or bubble discharging, and the plunger pump with smaller volume is used for the step of forming liquid drops.

20. A liquid droplet generation method according to claim 1, characterized in that: the first liquid is a continuous phase and the second liquid is a dispersed phase; and/or the first liquid is an oil phase and the second liquid is an aqueous phase.

21. A liquid droplet generation method according to claim 1 or 20, characterized in that: a surfactant is added into the first liquid; the second liquid is an aqueous phase containing the biological or chemical substance to be detected.

22. A liquid droplet generation method according to claim 1, characterized in that: the liquid drop is a digital PCR liquid drop or a single-cell liquid drop; and/or the diameter of the droplets is 50 micrometers to 250 micrometers, preferably 200 micrometers or less, more preferably 150 micrometers or less, further preferably 120 micrometers or less, and still further preferably 110 micrometers or less.

23. A droplet generation system comprising a droplet generation device and a droplet receptacle for receiving a first liquid and a droplet, characterized in that: the liquid drop generating device comprises an accommodating cavity with a variable volume, a control mechanism for controlling the volume of the accommodating cavity to be changed periodically, and a liquid drop generating pipe with a first port and a second port which are relatively far away, wherein the first port of the liquid drop generating pipe is communicated with the accommodating cavity, the liquid drop generating device further comprises a driving fluid mechanism for introducing driving fluid into the accommodating cavity, and the inner diameter of the second port of the liquid drop generating pipe is more than 0.1 millimeter.

24. A droplet generation system according to claim 23, wherein the second port of the droplet generation tube has an inner diameter greater than 0.2 mm and equal to or less than 1 mm; and/or the inner diameter of the first port is larger than the inner diameter of the second port; and/or the volume of the liquid drop generating pipe is 10-200 microliter.

25. A droplet generation system according to claim 24, wherein the second port of the droplet generation tube has an internal diameter of 0.3-0.6 mm.

26. A droplet generation system according to claim 23, wherein the periodic variation is a compression-recovery reciprocation variation, or an expansion-recovery reciprocation variation, or a compression-recovery-expansion-recovery reciprocation variation.

27. A liquid droplet generating system according to claim 23, wherein the driving fluid mechanism includes a pump and a fluid passage, the liquid droplet generating apparatus includes a base body which provides a cylindrical hole and the fluid passage and a connecting portion for connecting the liquid generating tube, the number of the cylindrical hole, the number of the fluid passage and the number of the connecting portion are one or more, the cylindrical hole is a cylinder shape which is open at the upper and lower sides, a diaphragm is provided on each of the cylindrical holes, and the cylindrical hole and the diaphragm together constitute the housing chamber.

28. A droplet generation system according to claim 27, wherein: the containing cavity, the liquid drop generating pipe and the liquid drop receiver are sequentially arranged from top to bottom, the first port of the liquid drop generating pipe is communicated with the lower opening of the containing cavity, and the central line of the containing cavity, the axial line of the liquid drop generating pipe, the central line of the first port and the central line of the second port are overlapped and extend along the vertical direction.

29. A droplet generation system according to claim 27, wherein: each diaphragm comprises a main body part and a movable part, the main body part is fixedly connected with the base body, the movable part is positioned right above the cylindrical hole, and the movable part is connected to the control mechanism through a connecting piece.

30. A droplet generation system according to any of claims 27 to 29, wherein: the diaphragm is a metal or non-metal diaphragm; and/or the thickness of the membrane is 0.005-2 mm; and/or a sealing structure is arranged between the membrane and the base body to seal the accommodating cavity.

31. A droplet generation system according to any of claims 27 to 29, wherein: the control mechanism is a vibration mechanism.

32. A droplet generation system according to claim 31, wherein: the vibration mechanism comprises one or more of a galvanometer motor, piezoelectric ceramics and a voice coil motor; and/or the vibration direction provided by the vibration mechanism is the up-down direction.

33. A droplet generation system according to claim 27, wherein: the liquid drop generating pipe is detachably connected with the connecting part; and/or, on the same base body, the number of the cylindrical holes, the number of the fluid passages and the number of the connecting parts are respectively 2-20.

34. A droplet generation system according to claim 27 or 33, wherein: the cylindrical holes, the fluid passages and the connecting parts are all multiple, two opposite side parts of the base body are higher than the middle part between the two opposite side parts respectively, the cylindrical holes are independently distributed in the middle part of the base body and are arranged in two rows, and each fluid passage comprises a vertical passage formed on the two opposite side parts of the base body and a horizontal passage for correspondingly communicating the vertical passage with the cylindrical holes.

35. A droplet generation system according to claim 34, wherein: a drainage part is formed between the port of the horizontal passage and the inner peripheral side wall of the accommodating cavity, so that liquid from the horizontal passage enters the accommodating cavity in a tangential mode along the circumferential direction of the accommodating cavity.

36. A droplet generation system according to claim 27, wherein: one end portion of the fluid passage communicates with the housing chamber, and when a fluid is driven from the fluid passage into the housing chamber, the fluid forms a vortex in the housing chamber and the droplet generation tube that rotates in the circumferential direction of the housing chamber and the droplet generation tube.

37. A droplet generation system according to claim 27, wherein: one end portion of the fluid passage communicates with the housing chamber, and a direction in which the fluid is discharged from the fluid passage is offset from an axial center line of the housing chamber.

38. A droplet generating apparatus, characterized in that: the method of any one of claims 23 to 37.

39. Use of a droplet generation method according to any of claims 1 to 22, a droplet generation system according to any of claims 23 to 37 and a droplet generation device according to claim 38 in clinical diagnostics, gene expression analysis, microbiological testing.

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