Droplet generation device, system and method for generating droplets

Abstract

The present invention relates to a droplet generation device, system, droplet generation method and their applications. The droplet generation device includes a volume-variable accommodating chamber, a control mechanism for controlling the volume change of the accommodating chamber, and a droplet generation tube having a first port and a second port that are relatively far apart, the first port of the droplet generation tube is connected to the accommodating chamber, and the inner diameter of the second port is above 0.2 mm. The droplet generation method includes: inserting the droplet generation tube into a first liquid so that the second port of the droplet generation tube is located below the liquid surface of the first liquid; controlling the volume of the accommodating chamber to change, and driving the second liquid to move, droplets are formed in the droplet generation tube and then flow out from the second port and enter the droplet receiver. The present invention provides a brand-new droplet generation method, which breaks through the limitation that the existing nanoliter-level droplet generation technology must use micro-pipes below 0.1 mm, and can achieve the preparation of small-volume uniform droplets at a significantly reduced cost.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2021113351133 and name “Droplet generation method, system and application thereof”. Technical Field

[0002] The present invention belongs to the technical field of droplet generation, and specifically relates to a new droplet generation method and a system used for the method, and also relates to the application of the droplet generation method and the droplet generation system in the fields of clinical diagnosis, gene expression analysis, microbial detection, etc. Background Art

[0003] In the field of biomolecular diagnosis, polymerase chain reaction (PCR) is used to extract and purify nucleic acid (DNA / RNA template) from samples and mix the probe primers to divide the liquid into uniform droplets; each droplet contains several or 0 templates, and the signal is amplified and detected after the PCR reaction, and the absolute quantification of the sample pathogen is achieved through Poisson distribution calculation, i.e. digital PCR. Digital PCR detection methods have important applications in gene mutation detection and analysis, targeted drug concomitant detection, cancer marker detection, next-generation gene sequencing and other fields.

[0004] With the development of science and technology, the combination of microfluidics and microdroplets has provided a new direction for some microreactions and microextractions. In these applications, the stable and uniform preparation of microdroplets is particularly important. Traditional droplet preparation techniques mostly use microchannels to form oil-in-water microdroplets by using the shear force between the oil phase and the liquid phase, or use physically cut micropores to separate droplets.

[0005] Referring to the patent with publication number CN 112439470 A, a dosing needle for preparing micro-droplets and a method for preparing micro-droplets are disclosed, wherein the dosing needle adopts a design of "an adapter for connecting a liquid supply adapter is provided at the upper part, a liquid storage part for sample storage is provided at the middle part, and a liquid discharge part for generating micro-droplets is provided at the lower part, the upper end opening of the adapter is a liquid supply opening, the lower end opening of the liquid discharge part is a liquid discharge opening, the diameters decrease successively from the liquid supply opening to the liquid discharge opening, the inner diameter of the liquid discharge opening is 25 to 200 microns, and the outer diameter is 200 to 800 microns, and when the dosing needle contacts the sample solution and the oily liquid, the contact angle is not less than 80°". When the dosing needle is used to prepare micro-droplets, the dosing needle performs periodic reciprocating motion with a varying speed in the oily liquid, so that the sample solution is subjected to periodic shear force of the oily liquid at the liquid discharge opening, and then the sample solution in the dosing needle enters the oily liquid, thereby generating micro-droplets. Obviously, this patent has strict requirements on the inner diameter, thickness, taper, angle, etc. of the sample adding tube, and the processing cost is high.

[0006] Refer to the patent with publication number CN 111841439 A, which discloses a device and method for preparing uniform single emulsion droplets with high throughput. When preparing droplets, the driving phase liquid is first continuously introduced into the cavity to form a stable driving phase flow field, and then the dispersed phase liquid is continuously introduced into the inlet end of the dispersed phase liquid channel. The actuating unit inputs disturbance to the dispersed phase liquid in the dispersed phase liquid channel through the disturbance interface to achieve control of the dispersed phase liquid jet breakup. The dispersed phase liquid is discharged from the outlet end of the capillary structure, wrapped by the driving phase liquid, and discharged from the discharge hole to form a single emulsion droplet. When preparing droplets in this patent, both 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 droplets need to be formed in the air when collected, which is contaminated by external factors and is not conducive to collection and subsequent analysis. Further, from its structural characteristics, it can be seen that this droplet generation method is not suitable for droplet preparation in biological detection fields such as digital PCR droplets or single-cell droplets. The reason is that in these fields, the amount of samples to be tested is usually small, which is not enough to form a stable and controllable jet as described in this patent. In addition, the structure of the device itself is relatively complex and the cost is very high. Summary of the invention

[0007] In order to overcome the problems existing in the preparation of existing droplets such as digital PCR droplets and single cell droplets, the present invention provides a novel droplet generation method.

[0008] The present invention further provides a novel droplet generation device and a droplet generation system, and further provides their applications in the fields of clinical diagnosis, gene expression analysis, microbial detection, etc.

[0009] In order to solve the above technical problems, a technical solution adopted by the present invention is:

[0010] A droplet generation method, the method using a droplet generation device and a droplet receiver, a first liquid is placed in the droplet receiver, the droplet generation device comprises a fluid passage, a volume-variable receiving chamber, and a droplet generation tube, the droplet generation tube has a first port and a second port that are relatively far apart, wherein the first port is connected to the receiving chamber, and the droplet generation method comprises the following steps:

[0011] S1, transferring a second liquid into the droplet generation tube, wherein the second liquid is a liquid that is immiscible with the first liquid;

[0012] S2, inserting the droplet generation tube into the first liquid so that the second port of the droplet generation tube is located below the liquid surface of the first liquid;

[0013] S3. Control the containing chamber to make its volume change periodically, and inject driving fluid into the fluid passage to drive the second liquid to move.

[0014] According to further contents of the droplet generation method of the present invention, in step S3, the droplets are formed in the droplet generation tube and then flow out of the second port and enter the droplet receiver. This is very unique. In contrast, the droplet generation method in the prior art always forms droplets outside the microchannel.

[0015] According to further contents of the droplet generation method of the present invention, in step S3, the droplet generation tube and the droplet receiver only need to remain relatively still, and there is no need to drive the droplet generation tube to make it vibrate back and forth as in the prior art.

[0016] According to further contents of the droplet generation method of the present invention, in step S3, the periodic change of the containing chamber is manifested as a change with a certain regularity, which can be a reciprocating change of compression-recovery, or a reciprocating change of expansion-recovery, or a reciprocating change of compression-recovery-expansion-recovery. In a specific embodiment, the periodic change is that the volume of the containing chamber decreases from V0 to V1, and then recovers from V1 to V0, and so on, repeatedly.

[0017] According to the further content of the droplet generation method of the present invention, the inner diameter of the second port is not less than 0.1 mm. Preferably, the inner diameter of the second port is greater than 0.2 mm, and specifically preferably 0.2 mm-1 mm. More preferably, the inner diameter of the second port is 0.3-0.6 mm. This is very unique. In contrast, in order to obtain micro-droplets in the prior art, the inner diameter of the outlet of the micro-channel is always below 0.1 mm. This unique feature brings significant advantages to the scheme of the present invention. First, since the preparation of uniform droplets is less affected by the processing accuracy of the micro-channel, the uniform controllability of the droplets is significantly improved. Second, the processing and manufacturing costs are significantly reduced. This unique feature is due to the fact that the droplet generation principle of the present invention is completely different 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 0.1 mm, otherwise droplets cannot be generated, and exceeding 0.2 mm is preferred. Basically, when the inner diameter of the second port exceeds 0.2 mm, uniform small-volume droplets can be stably obtained under a fairly wide range of conditions, especially when it is 0.3 mm-0.6 mm. At this time, the applicable range of conditions is the widest.

[0018] Preferably, the inner diameter of the first port is larger than the inner diameter of the second port.

[0019] Preferably, the droplet generation tube includes a tapered tube portion, and the two ends of the tapered tube portion respectively form 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 the ratio of the difference between the inlet diameter of the tapered tube portion (the inner diameter of the first port) and the outlet diameter of the tapered tube portion (the inner diameter of the second port) to the length of the tapered tube portion. Based on the droplet generation method of the present invention, the taper of the tapered tube portion has an important influence on the generation of droplets, which is reflected in that the adjustment of the taper can make it possible to obtain stable droplets even at a higher periodic change frequency (such as a vibration frequency). Therefore, the design of the taper can expand the applicable frequency range of the device to meet a wider range of needs.

[0020] Preferably, the frequency of the periodic change is 10 Hz-1 KHz, preferably 50 Hz-600 Hz, further preferably 80 Hz-600 Hz, more preferably 100 Hz-600 Hz, further preferably 150 Hz-600 Hz, further preferably 150 Hz-500 Hz; particularly preferably 150 Hz-300 Hz.

[0021] According to another specific implementation and preferred aspect of the present invention, in step S3, the accommodating chamber, the droplet generating tube and the droplet receiver are arranged in sequence from top to bottom, the first port of the droplet generating tube is connected to the bottom of the accommodating chamber, 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 coincide, and all extend in the vertical direction.

[0022] According to another specific implementation and preferred aspect of the present invention, at least a portion of the wall constituting the accommodating cavity is a movable portion, and the movable portion moves outward or inward under the action of an external force, thereby increasing or decreasing the volume of the accommodating cavity.

[0023] Preferably, the movable portion is made of a metal or non-metal film.

[0024] Furthermore, the movable part is provided on one or more of the top or surrounding sides of the accommodating cavity. In a preferred embodiment, the movable part is provided on a single side wall of the accommodating cavity, and further preferably, the movable part is provided on the top side of the accommodating cavity to constitute the top wall of the accommodating cavity.

[0025] In some preferred embodiments, the movable part is connected to the vibration mechanism through a connection mechanism, so that in step S3, the vibration mechanism drives the movable part to vibrate synchronously to control the periodic change of the volume of the accommodating chamber. Preferably, the direction of the reciprocating vibration is up and down.

[0026] In other preferred embodiments, the vibration mechanism is abutted against the movable part, and the reciprocating vibration of the vibration mechanism is transmitted to the movable part, causing it to vibrate so as to control the volume of the accommodating chamber to change periodically. Preferably, the direction of the reciprocating vibration is up and down.

[0027] According to the present invention, the type of vibration mechanism can be, for example, those already used in the prior art, and the present invention has no special requirements for this. However, as a preferred embodiment of the present invention, when generating droplets, the vibration frequency that can be used is 10Hz-1KHz; the vibration amplitude that can be used is 5 microns-1000 microns. Further preferably, the vibration frequency is 50Hz-600Hz, further preferably, the vibration frequency is 80Hz-600Hz, further preferably, the vibration frequency is 100Hz-600Hz, further preferably, 150Hz-600Hz, further preferably, 150Hz-500Hz, further preferably, 150Hz-300Hz. In some specific embodiments, the frequency of vibration is 100-300Hz. The amplitude of the vibration is preferably 5 microns-600 microns, more preferably 5 microns-300 microns, further preferably 5 microns-100 microns, and further 5 microns-60 microns. In some embodiments, the amplitude of vibration is 5 microns-50 microns, and in other embodiments, the amplitude of vibration is 20 microns-60 microns.

[0028] According to a further preferred embodiment of the present invention, when the taper of the conical tube portion is 0.05-0.1, the vibration frequency is set to 100 Hz-600 Hz, and the vibration amplitude is 10 microns-300 microns. When the taper of the conical tube portion is 0.1-0.2, the vibration frequency is set to 100-300 Hz, and the vibration amplitude is 10 microns-600 microns.

[0029] Preferably, in step S3, the fluid is a liquid. Further, the injection speed is 2-200 μl / min, preferably 10-50 μl / min.

[0030] Preferably, the accommodating cavity is an annular cavity with an inner diameter of 4-6 mm. In addition, the inner peripheral side wall of the accommodating cavity is preferably arranged to extend in the vertical direction. Also preferably, the inner wall surface of the accommodating cavity is a smooth surface.

[0031] 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 between the two has no effect on the generation effect of the droplets.

[0032] According to a specific implementation and preferred aspect of the present invention, in step S1, the second liquid is sucked into the second port of the droplet generating tube, and then a portion of the first liquid is sucked in, or the first liquid is not sucked in.

[0033] In some embodiments, the second liquid is sucked into the second port of the droplet generation tube, and then a portion of the first liquid is sucked in, so that before starting step S3, the liquid in the droplet generation tube is divided into a driving fluid segment, a second liquid segment, and a first liquid segment from top to bottom. The advantage of this is that the uniformity of the droplets prepared throughout the process can be improved.

[0034] In some embodiments, after the second liquid is sucked from the second port of the droplet generation tube, the first liquid is no longer sucked. Thus, before starting step S3, the liquid in the droplet generation tube is divided into a driving fluid segment and a second liquid segment from top to bottom.

[0035] According to another specific implementation and preferred aspect of the present invention, the droplet generation method further includes the step of cleaning and / or removing bubbles from the receiving chamber and the droplet generation tube after one droplet generation ends or before the next droplet generation begins.

[0036] Preferably, two plunger pumps of different volumes are used to control the driving fluid, and a three-way valve is used for switching control, wherein the larger plunger pump is used in the cleaning and / or bubble removal steps, and the smaller plunger pump is used in the droplet formation step.

[0037] According to the present invention, the first liquid is usually a continuous phase, and the second liquid is usually a dispersed phase; and / or, the first liquid is usually an oil phase, and the second liquid is usually an aqueous phase.

[0038] According to another specific implementation and preferred aspect of the present invention, a surfactant is added to the first liquid; and the second liquid is an aqueous phase containing biological or chemical substances to be detected.

[0039] Preferably, the droplet is a digital PCR droplet or a single cell droplet. The droplet has a diameter of 50 micrometers to 250 micrometers, preferably less than 200 micrometers, more preferably less than 150 micrometers, further preferably less than 120 micrometers, and further preferably less than 110 micrometers.

[0040] Another technical solution adopted by the present invention is: a droplet generation system, including a droplet generation device and a droplet receiver, the droplet receiver is used to accommodate a first liquid and droplets, the droplet generation device includes a accommodating chamber with a variable volume, a control mechanism for controlling the volume of the accommodating chamber to make it change periodically, and a droplet generation tube having a first port and a second port that are relatively far apart, the first port of the droplet generation tube is connected to the accommodating chamber, the droplet generation device also includes a driving fluid mechanism for introducing a driving fluid into the accommodating chamber, and the inner diameter of the second port of the droplet generation tube is above 0.1 mm.

[0041] As described above, the inner diameter of the second port is preferably greater than 0.2 mm, and more preferably 0.2 mm-1 mm. More preferably, the inner diameter of the second port is 0.3-0.6 mm. Also preferably, the inner diameter of the first port is greater than the inner diameter of the second port. The droplet generation tube includes a tapered tube portion, and the two ends of the tapered tube portion respectively form the first port and the second port, and the taper of the tapered tube portion is 0.05-0.2.

[0042] Furthermore, the volume of the droplet generation tube may be 10-200 microliters. Experiments show that the volume of the droplet generation tube has little effect on the droplet generation effect, which is also one of the advantages of the droplet generation method of the present invention.

[0043] According to a specific implementation and preferred aspect of the present invention, the periodic change is a reciprocating change of compression-recovery, or a reciprocating change of expansion-recovery, or a reciprocating change of compression-recovery-expansion-recovery.

[0044] According to a specific implementation and preferred aspect of the present invention, the driving fluid mechanism includes a pump and a fluid passage, and the droplet generating device includes a substrate, the substrate provides a cylindrical hole and a fluid passage, and provides a connecting portion connected to a liquid generating tube, and on the same substrate, the number of the cylindrical hole, the fluid passage, and the connecting portion is one or more, the cylindrical hole is a cylinder that is open at both the top and the bottom, and a diaphragm is covered on each cylindrical hole, and the cylindrical hole and the diaphragm together constitute a containing chamber.

[0045] Preferably, the accommodating chamber, the droplet generating tube and the droplet receiver are arranged in sequence from top to bottom, the first port of the droplet generating tube is connected to the lower opening of the accommodating chamber, 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 coincide, and all extend in the vertical direction.

[0046] Preferably, each diaphragm comprises a main body and a movable part, the main body is fixedly connected to the substrate, the movable part is located directly above the cylindrical hole, and the movable part is connected to the control mechanism through a connecting piece.

[0047] Furthermore, the diaphragm may be a metal (such as stainless steel) or a non-metal diaphragm; the thickness of the diaphragm may be 5 micrometers to 2 millimeters.

[0048] Preferably, a sealing structure is provided between the diaphragm and the base body so that the accommodating cavity always maintains a good seal.

[0049] Preferably, the control mechanism is a vibration mechanism, and the vibration mechanism includes one or more of a galvanometer motor, a piezoelectric ceramic, and a voice coil motor, without particular limitation. According to a preferred aspect of the present invention, the vibration mechanism preferably includes a piezoelectric ceramic.

[0050] Preferably, the vibration direction provided by the vibration mechanism is the up and down direction, and the vibration frequency provided includes at least 50-600 Hz. When used, it can be specifically selected within this range, and the vibration amplitude provided preferably includes at least 5 microns-300 microns.

[0051] Preferably, the droplet generation 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 disassembled and replaced when the droplet generation of the sample solution is completed. In some embodiments, the droplet generation tube can directly use a commercially available nozzle of corresponding specifications. Of course, it is also possible to make the droplet generation tube and the connecting portion into one piece, which will not affect the droplet generation effect.

[0052] Preferably, on the same substrate, the number of the cylindrical holes, fluid passages and connecting parts is 2 to 20, preferably 4 to 10.

[0053] Preferably, there are multiple cylindrical holes, fluid passages, and connecting parts, and the two opposite sides of the base are respectively higher than the middle part between the two opposite sides. The multiple cylindrical holes are independently distributed in the middle part of the base and arranged in two rows. Each fluid passage includes a vertical passage formed on the two opposite sides of the base and a horizontal passage correspondingly connecting the vertical passage with the cylindrical hole. With this structural design, the device is more compact as a whole and convenient to operate and control.

[0054] According to a specific implementation and preferred aspect of the present invention, a drainage portion is formed between the port of the horizontal passage and the inner peripheral side wall of the accommodating chamber, so that the liquid from the horizontal passage enters the accommodating chamber in a tangential direction to the circumference of the accommodating chamber.

[0055] According to a specific implementation and preferred aspect of the present invention, one end of the fluid passage is connected to the receiving chamber of the substrate, and when the fluid is driven from the fluid passage into the receiving chamber, the fluid forms a vortex in the receiving chamber and the droplet generating tube that rotates circumferentially along the receiving chamber and the droplet generating tube.

[0056] According to another specific implementation and preferred aspect of the present invention, one end of the fluid passage is communicated with the accommodating cavity of the substrate, and the direction in which the fluid is discharged from the fluid passage deviates from the axial centerline of the accommodating cavity.

[0057] As described above, through the structural design of the fluid passage and the containing cavity, the bubbles in the system cavity can be easily discharged before droplet generation, thereby avoiding the adverse effect of the bubbles on droplet generation.

[0058] According to the present invention, the droplet receiver is a device having a containing cavity, which may be in a sealed or non-sealed form without particular limitation.

[0059] Another technical solution adopted by the present invention is: a droplet generating device, which has the same structure as the droplet generating device in the above droplet generating system, but does not include a droplet receiver.

[0060] Another technical solution of the present invention is: an application of the above-mentioned droplet generation method, droplet generation system and droplet generation device in clinical diagnosis, gene expression analysis and microbial detection.

[0061] In the present invention, there is no particular limitation on the oil phase, which can be any suitable oil phase reported in the prior art. Preferably, the oil phase includes oil and surfactant. Specifically, the oil can be a combination of one or more of fluorocarbon oil, silicone oil, mineral oil, hydrocarbon oil, and vegetable oil. The surfactant generally has a hydrophilic-lipophilic balance (HLB) of 3 to 6. The content of the surfactant in the oil phase is generally 0.1wt% to 20wt%, preferably 0.1% to 10%.

[0062] A large number of surfactants suitable for generating water-in-oil droplets have been reported in the prior art, and they are all suitable for use in the oil phase of the present invention.

[0063] As a specific and preferred aspect of the present invention, when the oil is a fluorocarbon oil, the surfactant is a fluorine-containing surfactant, such as perfluorooctanol, perfluorodecanol, perfluorotetradecanoic acid, perfluoropolyether carboxylic acid and derivatives thereof.

[0064] As another specific and preferred aspect of the present invention, when the oil is silicone oil, the surfactant can be a nonionic surfactant such as polyethylene glycol octylphenyl ether (TritonX-100), a silicon-oxygen chain nonionic surfactant such as ABIL EM90, an anionic surfactant such as SDS, etc.

[0065] As another specific and preferred aspect of the present invention, when the oil is a hydrocarbon oil, the surfactant can be a nonionic surfactant such as Tween 20, Tween 80, a silicon chain nonionic surfactant such as ABIL EM90, an anionic surfactant such as SDS, phospholipids, etc.

[0066] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0067] The present invention provides a new droplet generation method, which breaks through the limitation that the existing nanoliter-scale droplet generation technology must use micro-channels below 0.1 mm, and can achieve the preparation of small-volume uniform droplets at a significantly reduced cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0069] Figure 1 Schematic diagram of the structure of the droplet generating device of Example 1;

[0070] Figure 2 for Figure 1 Schematic diagram of the local structure;

[0071] Figure 3 for Figure 2 Schematic diagram of the local structure;

[0072] Figure 4 for Figure 3 A cross-sectional schematic diagram of

[0073] Figure 5 is a schematic cross-sectional view of a single droplet generating unit in the droplet generating device of Example 1;

[0074] Figure 6 for Figure 5 AA section view;

[0075] Figure 7 It is a schematic cross-sectional view of the structure of the droplet generation system of Example 1;

[0076] Figure 8 is a cross-sectional schematic diagram of a single droplet generation unit filled with driving oil in Example 1;

[0077] Fig. 9 It is a cross-sectional schematic diagram of forming a driving fluid segment and a second liquid segment in a single droplet generation unit in Example 1;

[0078] Fig.10 It is a schematic cross-sectional view of the structure of the droplet generation system of Example 2;

[0079] Figures 11 to 16 Microscope images of the droplets prepared in Examples 3 to 7, respectively;

[0080] Fig.17 A schematic diagram showing the velocity field distribution near the outlet of the droplet generation tube;

[0081] In the accompanying drawings: 1. droplet generating device; 10. substrate; 10a. middle part; 10b. left raised edge part; 10c. right raised edge part; 100. cylindrical hole; 101. connecting part; t. channel; 11. droplet generating unit; 110. accommodating chamber; 111. control mechanism; 112. droplet generating tube; a1. first port; a2. second port; c1. connecting tube part; c2. conical tube part; 113. driving fluid mechanism; d1. pump; d2. fluid passage; d21. vertical passage; d22. horizontal passage; d3. drainage part; 114. diaphragm; b1. main body; b2. movable part; 115. connecting piece; y1. first liquid; y2. second liquid; y3. fluid; 116. sealing ring; 2. droplet receiver. DETAILED DESCRIPTION

[0082] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0083] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0084] In the prior art, it is generally believed that the smaller the microchannel used to generate microdroplets, the better. Therefore, the inner diameter of the microchannel actually used is usually less than 0.1 mm. At the same time, the microdroplet generation technology in the prior art mainly includes the co-flow focusing method and the microchannel vibration method. For the co-flow focusing method, the dispersed phase liquid is vibrated and sprayed into the continuous phase and is wrapped by the continuous phase, and then sprayed out from the spray hole together to form droplets. For the microchannel vibration method, the microchannel (sample tube) itself needs to be reciprocated to generate droplets.

[0085] The inventors of the present invention have found in a large number of experimental studies that the preparation of uniform droplets can be achieved by simply combining the vibration of the dispersed phase and the micro-channels of appropriate inner diameter. Based on this discovery, the inventors further studied its mechanism, conducted a simulation structural analysis of the key factors affecting the generation of droplets, and verified it through further experiments. Studies have shown that the droplet generation of the present invention has a new generation principle that is significantly different from any droplet generation method in the prior art. Compared with the existing droplet generation method, the droplet generation method can truly realize the generation of nanoliter volume droplets from non-microfluidic scale structures (all the geometric scales of the cavities and consumables related to the generation process disclosed in the present invention are above 0.1 mm; generally speaking, 0.1 mm is the key dimension to distinguish microfluidics). However, the structural scales that can actually be used in the droplet generation technology in the fields of digital PCR, single cell sorting, etc. in the prior art are all less than or close to the diameter of 0.1 mm of the nanoliter droplet. Using this droplet generation technology, nano-scale droplets can be generated with structures that are much larger than the size of nano-droplets, which is a core technological breakthrough in achieving the cost of using droplet digital PCR. Based on this droplet generation technology, even a general pipette tip can be directly used as a key generation consumable. This droplet generation technology has no other mechanical movement except the micro-movement of the dispersed phase. We name this technology non-vibrational ejection technology. The present invention discloses a technology for generating droplets by causing the dispersed phase to become unstable through the velocity gradient in the droplet generation pipeline, achieving nanoliter precision control.

[0086] The droplet generation principle of the present invention is described as follows:

[0087] The driving mechanism (such as a vibration mechanism) pushes and pulls the movable part through direct connection with the movable part (such as an elastic diaphragm) or touches the movable part through contact with the movable part, so that the movable part generates periodic vibrations, driving the liquid (second liquid / dispersed phase / water phase) in the accommodating cavity to generate periodic motion, and at the same time continuously injecting the driving fluid (driving oil) into the cavity, a periodic motion including forward push, ejection and retraction motion is generated at the oil-water interface (the interface between the first liquid and the second liquid) at the outlet (second port) of the droplet generation tube. In the forward push, ejection stage, according to the characteristics of the tubular fluid, it can be seen from the Poiseuille flow that the flow velocity in the middle of the tube will be greater than the flow velocity near the wall (such as Fig.17As shown, the velocity field distribution at the outlet of the droplet generation tube is shown, and the brightness indicates the velocity. It can be seen that the velocity at the center of the outlet is the largest, which will stretch the oil-water interface), resulting in the movement speed of the middle interface being greater than the movement speed of the interface close to the wall, thereby causing the oil-water interface to form a conical interface, and the interface is continuously elongated. Since the surface tension at the oil-water interface has a tendency to shrink, the interface tends to be cut into droplets. When the stretched interface gradually becomes slender, according to the Laplace equation, the interfacial tension gradually increases, and the interface can be cut when it exceeds a certain critical value, and Rayleigh Taylor instability occurs to form droplets. This is also the key reason why the droplet generation tube outlet can still be generated when it is much larger than the droplet diameter (~0.1mm). The subsequent retraction movement will pull back the conical interface to complete a cycle of movement. Another point that needs to be pointed out is that the study found that the exact location of droplet generation is in the droplet generation pipeline (in some specific experiments, droplets are generated at about 0.5mm from the outlet), which is different from other generation technologies where droplets are formed outside the pipeline outlet. This also reflects the uniqueness of the technology of the present invention.

[0088] The vibration-free ejection technology based on the present invention has obvious advantages over the droplet generation method using micro-channels to continuously vibrate at high frequency in oily liquids. On the one hand, there is no strict requirement for the depth of the micro-channels extending into the oil phase liquid, and it will not damage the droplets that have been generated. The quality of the droplets in the overall generation process and the operation of the generation are more controllable; on the other hand, the requirements for the inner diameter of the micro-channels in the prior art are all within 0.1 mm. The larger the inner diameter, the higher the high-frequency oscillation frequency required to be applied, the higher the control requirements, the poorer the stability, and the higher the consistency requirements for the micro-channels themselves during manufacturing. The present invention applies vibration to the aqueous phase liquid to achieve the generation of droplets, and the requirements for the inner diameter of the liquid inlet and outlet of the micro-channel are reduced (can be greater than 0.1 mm, preferably more than 0.3 mm), which can ensure the uniformity of the droplets and make the control easier, significantly reducing the difficulty of processing the sample tube and reducing the processing cost. Compared with the co-flow focusing method, the present invention can greatly simplify the structure of the system under the premise of achieving the uniformity and stability of droplet generation, and the structure is simpler, the operation is more convenient, and the cost is significantly reduced.

[0089] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0090] Example 1

[0091] like Figures 1 to 9 As shown, the droplet generation system involved in this embodiment includes a droplet generation device 1 and a droplet receiver 2.

[0092] The droplet generating device 1 comprises a substrate 10 and a plurality of droplet generating units 11 arranged on the substrate 10 , wherein the plurality of droplet generating units 11 are arranged in parallel, and single-channel or multi-channel droplet generation can also be realized simultaneously.

[0093] Combination Figure 2 As shown, the substrate 10 is an aluminum block in the shape of an elongated strip, and the middle portion of the elongated strip is recessed downward from the surface to form an intermediate portion 10a, a left raised edge portion 10b, and a right raised edge portion 10c, wherein a plurality of droplet generating units 11 are arranged side by side and evenly spaced along the length direction of the substrate 10.

[0094] Combination Figure 3 and Figure 4 As shown, eight cylindrical holes 100 extending up and down are provided on the middle portion 10a of the base body 10, and a connecting portion 101 is provided correspondingly below each cylindrical hole 100. The eight cylindrical holes 10 are divided into two rows, wherein each row has four cylindrical holes 10 and are evenly spaced, and the left and right rows of cylindrical holes 10 are aligned left and right.

[0095] Combination Figure 5 As shown, each droplet generating unit 11 includes a accommodating chamber 110 with a variable volume, a control mechanism 111 for controlling the volume of the accommodating chamber 110 to make it change periodically, a droplet generating tube 112 having a first port a1 and a second port a2 that are relatively far apart, and a driving fluid mechanism 113 for introducing a driving fluid into the accommodating chamber 110.

[0096] In this example, a diaphragm 114 is covered on the cylindrical hole 100, and the cylindrical hole 100 and the corresponding diaphragm 114 together form a receiving chamber 110. The control mechanism 111 is a vibration mechanism, which is installed above the cylindrical hole 100 and connected to the diaphragm 114 through a connector 115, so as to control the movement of the diaphragm 114 to implement the volume change of the receiving chamber 110. The droplet generation tube 112 is vertically arranged, wherein the upper end is connected to the connecting part 101, and the lower end forms a droplet outlet. The driving fluid mechanism 113 is arranged at the left raised edge portion 10b or the right raised edge portion 10c close to the cylindrical hole 100.

[0097] The center line of the accommodating chamber 110, the axis of the droplet generating tube 112, the center line of the first port a1, and the center line of the second port a2 coincide with each other, and all extend in the vertical direction. The accommodating chamber 110 is an annular cavity with an inner diameter of about 5 mm, and the inner peripheral side wall extends in the vertical direction. The diaphragm 114 (stainless steel material) constituting the top of the accommodating chamber 110 includes a main body b1 and a movable part b2, wherein the main body b1 is fixedly connected to the substrate 10, the movable part b2 is located directly above the cylindrical hole 100, and the movable part b2 is connected to the control mechanism 111 through a connector 115. The control mechanism 111 specifically includes piezoelectric ceramics, which can provide up and down reciprocating vibration. When it reciprocates, it will drive the movable part b2 to move inward or outward relative to the accommodating chamber 110, thereby correspondingly changing the volume of the accommodating chamber 110 periodically, and accordingly, the liquid in the accommodating chamber 110 is disturbed by the periodic volume change. Further, the periodic change can be a reciprocating change of compression-recovery, or a reciprocating change of expansion-recovery, or a reciprocating change of compression-recovery-expansion-recovery. In this example, at least the periodic change that can be provided includes the reciprocating change of compression-recovery. The further piezoelectric ceramic is 40VS12 (having an internal thread connected to the connector 115).

[0098] The droplet generation tube 112 includes a connecting tube portion c1 and a tapered tube portion c2. The tapered tube portion c2 has a first port a1 and a second port a2. The inner diameter of the tapered tube portion c2 gradually decreases from the first port a1 to the second port a2, and the taper presented by the change in the inner diameter has an important influence on the droplet generation effect. Assume that the inner diameter of the first port a1 is R1, the inner diameter of the second port a is R2, the distance between the first port a1 and the second port a2 (that is, 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 used is 0.12, and the inner diameter R2 of the second port a2 is 0.5±0.1mm. The volume of the droplet generation tube 112 is about 10 microliters.

[0099] The connecting tube c1 intersects with the tapered tube c2 at the first port a1, and is used to be detachably sleeved on the connecting part 101. There is no special requirement for its taper, but it is preferably greater than the taper of the tapered tube c2. A flow channel t connected to the accommodating chamber 110 is formed in the middle of the connecting part 101. After the droplet generation tube 112 is installed on the connecting part 101, the accommodating chamber 110 is connected to the droplet generation tube 112 through the flow channel t.

[0100] Combination Figure 6As shown, the fluid driving 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 connects the vertical passage d21 to the cylindrical hole 100. In order to help discharge the bubbles in the system cavity, a drainage portion d3 is formed between the port of the horizontal passage d22 and the inner peripheral side wall of the receiving cavity 110, so that the liquid from the horizontal passage d2 enters the receiving cavity 110 in a direction tangent to the circumference of the receiving cavity 110, so that the fluid forms a vortex that rotates along the circumference of the receiving cavity 110 and the droplet generating tube 112 in the receiving cavity 110 and the droplet generating tube 112, thereby easily discharging the bubbles. In other embodiments, the provision of the drainage portion is not necessary, as long as the direction in which the fluid is discharged from the fluid passage d22 deviates from the axis of the receiving cavity, the bubble discharge effect can be better achieved.

[0101] In this example, two plunger pumps of different volumes are used to control the driving fluid, and a three-way valve is used for switching control. The larger plunger pump is used in the cleaning and / or bubble removal steps, and the smaller plunger pump is used in the droplet formation step.

[0102] In this example, a sealing ring 116 is further provided between the diaphragm 114 and the base body 10 to improve the sealing performance of the accommodating cavity.

[0103] Combination Figure 7 As shown, the droplet receiver 2 is located below the second port a2 and is used to receive the first liquid y1 and the droplets.

[0104] In the field of digital PCR, the first liquid y1 is usually a formulated oil, such as mineral oil, preferably with a surfactant added thereto. The second liquid y2 is usually an aqueous phase of the biological or chemical substance to be detected.

[0105] Combination Figure 8 As shown, the fluid y3 (driving oil) fills the fluid passage d2, the accommodating chamber 110 and the inner cavity of the droplet generating tube 112. Furthermore, the fluid y3 and the first liquid y1 can be made of the same mineral oil.

[0106] Combination Fig. 9 As shown, generally, 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 top to bottom. Alternatively, the liquid in the droplet generation tube 112 is divided into a driving fluid segment, a second liquid segment, and a first liquid segment from top to bottom; then, the driving mechanism and the fluid driving mechanism are turned on to start generating droplets.

[0107] Example 2

[0108] like Fig.10As shown, the droplet generating device provided in this embodiment is basically the same as that in 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 corresponding to the tapered tube portion c2 of the droplet generating tube 112 is 0.06, that is, the taper of the tapered tube portion of the droplet generating tube 112 in this example is half of the corresponding taper of Embodiment 1.

[0109] Example 3

[0110] The droplet generation method of this embodiment adopts the droplet generation system involved in the embodiment, and the implementation process is as follows:

[0111] S1. Use a larger pump (plunger pump) to fill the fluid passage, the containing cavity and the inner cavity of the droplet generation tube with fluid (driving oil), then suck the water phase to be tested (second liquid) into the droplet generation tube through the second port, and then suck a section of formula oil (first liquid). In this way, the liquid in the liquid generation tube includes three sections, which are driving oil section, water phase section and formula oil section from top to bottom;

[0112] S2, inserting the droplet generation tube into the formulated oil so that the second port of the droplet generation tube is located about 2 mm below the liquid surface of the formulated oil;

[0113] S3. The movable part of the diaphragm is directly pushed and pulled to vibrate synchronously up and down under the vibration drive of the vibration mechanism, the accommodating chamber is controlled to make its volume change periodically, and driving oil is injected into the fluid passage to drive the movement of the water phase. At this time, droplets are generated in the conical tube portion of the droplet generation tube, and then the formed droplets flow out through the second port of the droplet generation tube and enter the droplet receiver. In this process, there is no need to drive the droplet generation tube to move, and the droplet generation tube and the droplet receiver remain relatively still.

[0114] In this example, according to the above steps, water is used as the second liquid, the injection speed of the driving oil is 36.5 μl / min, the vibration frequency of the piezoelectric ceramic is 150 Hz, the vibration amplitude is 50 μm, and the input voltage is 2.5 V. Fig.11 , Fig.12 As shown, it can be seen that droplets of very uniform size were successfully prepared, and the diameter of the droplets was between 104 microns and 106 microns.

[0115] Example 4

[0116] The steps of the droplet generation method of this embodiment are basically the same as those of Embodiment 3, except that the injection speed of the driving oil is changed.

[0117] The droplets were prepared under the conditions of an injection rate of the driving oil of 19.5 μl / min, a vibration frequency of the piezoelectric ceramic of 150 Hz, a vibration amplitude of 50 μm, and an input voltage of 2.5 V. The droplet diameter was as shown in Fig.13a As shown, the diameter of the generated droplets is about 85±1 microns.

[0118] The droplets were prepared under the conditions of an injection rate of the driving oil of 15.0 μl / min, a vibration frequency of the piezoelectric ceramic of 150 Hz, a vibration amplitude of 50 μm, and an input voltage of 2.5 V. The droplet diameter was as shown in Fig.13b As shown, the diameter of the generated droplets is about 78±1 microns.

[0119] Example 5

[0120] The steps of the droplet generation method of this embodiment are basically the same as those of Embodiment 3, except that the vibration frequency is changed.

[0121] The droplets were prepared under the conditions of an injection rate of the driving oil of 48.7 μl / min, a vibration frequency of the piezoelectric ceramic of 200 Hz, a vibration amplitude of 50 μm, and an input voltage of 2.5 V. The droplet diameter was Fig.14a As shown, the diameter of the generated droplets is between 104.00 and 107.00 microns.

[0122] The droplets were prepared under the conditions of an injection rate of the driving oil of 60.8 μl / min, a vibration frequency of the piezoelectric ceramic of 250 Hz, a vibration amplitude of 50 μm, and an input voltage of 2.5 V. The droplet diameter was as shown in Fig.14b As shown, the diameter of the generated droplets is between 103.00 and 108.50 microns.

[0123] The droplets were prepared under the conditions of an injection rate of the driving oil of 73.0 μl / min, a vibration frequency of the piezoelectric ceramic of 300 Hz, a vibration amplitude of 50 μm, and an input voltage of 2.5 V. The droplet diameter was as shown in Fig.14c and Fig.14d As shown, the diameter of the generated droplets is between 93.00 and 106.00 microns.

[0124] It can be seen from this embodiment that the droplet generation method of the present invention can obtain relatively uniform droplets at different frequencies.

[0125] Example 6

[0126] This embodiment uses the droplet generation device involved in Example 2, that is, a droplet generation tube with a smaller taper, and conducts experiments at different vibration frequencies. The results show that when the vibration frequency of the piezoelectric ceramic is 600 Hz, uniform droplets can still be obtained. The effect of preparing droplets at 150 Hz is equivalent to that of Example 3.

[0127] Example 7

[0128] The droplet generation method of this embodiment has the same steps as those of embodiment 3, except that RCR reagent is used instead of water as the second liquid to generate droplets. The RCR reagent uses a 20ul system: Bio-Rad supermix 10ul+Bio-Rad demo kit DNA 1ul+Fam probe 1ul+Hex probe 1ul+Water 7ul.

[0129] The prepared droplets are Fig.15 , Fig.16 As shown, it can be seen that droplets of very uniform size were successfully prepared, and the diameter of the droplets was about 104-107 microns.

[0130] In each of the above embodiments, in order to prevent cross contamination of different samples, the droplet generation tube can be disassembled and replaced after use. At the same time, the above droplet generation solution can also be applied in the following fields.

[0131] Quantification: 1) Analysis of genetically modified components. Microbiological testing: 1) Water sample microbiological testing; 2) Pathogenic microorganism testing.

[0132] It can be seen from the above embodiments that the present invention has multiple technical advantages in the preparation of droplets, including but not limited to:

[0133] 1. Uniform micro-droplets can be formed through a simple structure, which has technical advantages such as repeatable and continuous preparation. It can be used in clinical diagnosis, gene expression analysis, microbial detection and other occasions, and has good practicality;

[0134] 2. Droplets are generated in the droplet generation tube. Under the condition of a given device, the droplet generation effect is mainly affected by the vibration frequency, and is insensitive to slight changes in the inner diameter of the droplet generation tube, the position of the droplet generation tube inserted below the oil phase interface, the formula composition of the oil phase, etc. Therefore, the consistency and controllability of droplet generation are significantly improved;

[0135] 3. The device itself is equipped with a bubble removal structure and function, which can effectively remove bubbles during cleaning and avoid interference with droplet generation due to the presence of bubbles;

[0136] 4. The requirements for the inner diameter control of the droplet generation tube are significantly reduced, which can significantly reduce the cost accordingly. Therefore, the droplet generation tube can be used as a consumable to prevent mutual contamination between the generated samples, and a disposable droplet generation tube can be used;

[0137] 5. The prepared droplets are directly stored in the droplet receiver without transfer. The prepared droplets can be directly amplified and analyzed by PCR, realizing the integration of droplet generation and analysis;

[0138] 6. The above application can divide the sample into micro-droplets of uniform size for detection, which has the advantages of high specificity, high sensitivity and high accuracy of the detection results; at the same time, the detection of a single micro-droplet can also facilitate the analysis and research of the sample at the microscopic level;

[0139] 7. The droplet generation system and method of the present invention have a wide range of applications, including but not limited to the following:

[0140] Clinical diagnosis: 1) Non-invasive prenatal diagnosis: detection of fetal genetic diseases through free DNA fragments from the mother; 2) Cancer marker detection; 3) Virus detection; 4) Copy number variation analysis; 5) Mutation detection.

[0141] Gene expression analysis (mainly analyzing genetic differences between cells): 1) Gene expression analysis; 2) Single-cell gene expression analysis.

[0142] Next generation sequencing: 1) Sequencing result verification; 2) Sequencing library quality control.

[0143] Quantification of genetically modified components: analysis of genetically modified components.

[0144] Microbiological testing: 1) Microbiological testing of water samples; 2) Pathogenic microorganism testing.

[0145] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A droplet generating device, characterized in that: The droplet generating device comprises a accommodating chamber with a variable volume, a control mechanism for controlling the volume change of the accommodating chamber, and a droplet generating tube having a first port and a second port relatively far apart, the first port of the droplet generating tube being connected to the accommodating chamber, the inner diameter of the second port of the droplet generating tube being above 0.2 mm, the droplet generating device further comprises a driving fluid mechanism for introducing a driving fluid into the accommodating chamber, the driving fluid mechanism comprises a pump and a fluid passage; the droplet generating device comprises a substrate, the substrate provides a cylindrical hole and the fluid passage and provides a connecting portion for connecting the droplet generating tube, the cylindrical hole is open at both the upper and lower parts, a diaphragm is covered on the upper opening of the cylindrical hole, the cylindrical hole and the diaphragm together constitute the accommodating chamber, and the cylindrical hole, There are multiple fluid passages and connecting parts, the two opposite side parts of the substrate are respectively higher than the middle part between the two opposite side parts, and the multiple cylindrical holes are independently distributed in the middle part of the substrate and arranged in two rows. Each fluid passage includes a vertical passage formed on the two opposite side parts of the substrate and a horizontal passage correspondingly connecting the vertical passage with the cylindrical holes. A drainage part is formed between the port of the horizontal passage and the inner peripheral side wall of the receiving cavity, so that the liquid from the horizontal passage enters the receiving cavity in a tangential direction to the circumference of the receiving cavity; one end of the fluid passage is connected to the receiving cavity of the substrate, and when the fluid is driven to enter the receiving cavity from the fluid passage, the fluid forms a circumferentially rotating vortex of the receiving cavity and the droplet generating tube in the receiving cavity and the droplet generating tube, and the direction in which the fluid is discharged from the fluid passage deviates from the axial line of the receiving cavity.

2. The droplet generating device according to claim 1, characterized in that: The inner diameter of the second port of the droplet generating tube is less than or equal to 1 mm.

3. The droplet generating device according to claim 1, characterized in that: The inner diameter of the second port of the droplet generation tube is 0.3 mm to 0.6 mm.

4. The droplet generating device according to claim 1, characterized in that: An inner diameter of the first port is greater than an inner diameter of the second port.

5. The droplet generating device according to claim 1, characterized in that: The droplet generation tube includes a tapered tube portion, two ends of the tapered tube portion respectively form the first port and the second port, and the tapering of the tapered tube portion is 0.05-0.

2.

6. The droplet generating device according to claim 1, characterized in that: The volume of the droplet generation tube is 10-200 μl.

7. The droplet generating device according to claim 1, characterized in that: At least a portion of the wall constituting the accommodating chamber is a movable portion, which moves outward or inward under the action of an external force, thereby increasing or decreasing the volume of the accommodating chamber; the control mechanism is a vibration mechanism, and the volume of the accommodating chamber changes with the reciprocating vibration of the vibration mechanism.

8. The droplet generating device according to claim 7, characterized in that: The vibration direction of the vibration mechanism is the up and down direction. When the vibration mechanism moves from top to bottom, the volume of the accommodating chamber becomes smaller; when the vibration mechanism moves from bottom to top, the volume of the accommodating chamber becomes larger; when the vibration mechanism vibrates reciprocatingly up and down, the volume of the accommodating chamber changes periodically, and the periodic change is a reciprocating change of compression-recovery.

9. The droplet generating device according to claim 8, characterized in that: The vibration mechanism is a vibration mechanism capable of providing an amplitude of 5 micrometers to 1000 micrometers and a frequency of 10 Hz to 1 KHz.

10. The droplet generating device according to claim 8, characterized in that: The vibration mechanism includes one or more of a galvanometer motor, a piezoelectric ceramic, and a voice coil motor.

11. The droplet generating device according to any one of claims 1 to 10, characterized in that: The accommodating chamber and the droplet generating tube are arranged in sequence from top to bottom, the first port of the droplet generating tube is connected to the lower opening of the accommodating chamber, 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 coincide with each other, and all extend in the vertical direction.

12. The droplet generating device according to claim 1, characterized in that: The diaphragm comprises a main body and a movable part, wherein the main body is fixedly connected to the base, the movable part is located directly above the cylindrical hole, and the movable part is connected to the control mechanism through a connecting piece.

13. The droplet generating device according to claim 1, characterized in that: The diaphragm is a metal or non-metal diaphragm.

14. The droplet generating device according to claim 1, characterized in that: The thickness of the diaphragm is 0.005-2 mm.

15. The droplet generating device according to claim 1, characterized in that: A sealing structure is provided between the diaphragm and the base to seal the accommodating cavity.

16. A droplet generating device, characterized in that: The invention comprises a accommodating chamber with a variable volume, a vibration mechanism for controlling the volume of the accommodating chamber to make it change periodically, and a droplet generating tube having a first port and a second port relatively far apart, wherein the first port of the droplet generating tube is connected to the accommodating chamber, and the droplet generating device further comprises a driving fluid mechanism for introducing a driving fluid into the accommodating chamber, and the inner diameter of the second port of the droplet generating tube is greater than 0.2 mm; at least a part of the wall constituting the accommodating chamber is a movable part, and the movable part generates an outward or inward movement under the action of an external force, thereby increasing or decreasing the volume of the accommodating chamber; the droplet generating device further comprises a driving fluid mechanism for introducing a driving fluid into the accommodating chamber, and the driving fluid mechanism comprises a pump and a fluid passage; the droplet generating device comprises a substrate, and the substrate provides a cylindrical hole and the fluid passage and provides a connecting part for connecting the droplet generating tube, and the cylindrical hole is An opening is provided, and a diaphragm is covered on the upper opening of the cylindrical hole. The cylindrical hole and the diaphragm together constitute the accommodating chamber, and there are multiple cylindrical holes, fluid passages, and connecting parts. The two opposite side parts of the substrate are respectively higher than the middle part between the two opposite side parts. The multiple cylindrical holes are independently distributed in the middle part of the substrate and arranged in two rows. Each fluid passage includes a vertical passage formed on the two opposite side parts of the substrate and a horizontal passage correspondingly connecting the vertical passage with the cylindrical hole. A drainage part is formed between the port of the horizontal passage and the inner peripheral side wall of the accommodating chamber, so that the liquid from the horizontal passage enters the accommodating chamber in a tangential direction to the circumference of the accommodating chamber; one end of the fluid passage is connected to the accommodating chamber of the substrate, and when the fluid is driven to enter the accommodating chamber from the fluid passage, the fluid forms a circumferentially rotating vortex of the accommodating chamber and the droplet generating tube in the accommodating chamber and the droplet generating tube, and the direction in which the fluid is discharged from the fluid passage deviates from the axial line of the accommodating chamber.

17. The droplet generating device according to claim 16, characterized in that: The inner diameter of the second port of the droplet generation tube is 0.3 mm to 0.6 mm.

18. The droplet generating device according to claim 16, characterized in that: The movable part is made of a metal or non-metal film.

19. The droplet generating device according to claim 16, characterized in that: The movable part is connected to the vibration mechanism through a connecting piece.

20. The droplet generating device according to claim 16, characterized in that: The periodic change is a reciprocating change of compression-recovery, or a reciprocating change of expansion-recovery, or a reciprocating change of compression-recovery-expansion-recovery.

21. A droplet generation system for generating droplets with a diameter of less than 250 microns, characterized in that: The device comprises a droplet generating device and a droplet receiver according to any one of claims 1 to 20.

22. A method for generating droplets with a diameter of less than 250 microns, characterized in that: The method adopts the droplet generation device according to any one of claims 1 to 20 or the droplet generation system according to claim 21, and comprises the following steps: S0, adding the first liquid into the droplet receiver; S1, transferring a second liquid into the droplet generation tube, wherein the second liquid is a liquid that is immiscible with the first liquid; S2, inserting the droplet generation tube into the first liquid so that the second port of the droplet generation tube is located below the liquid surface of the first liquid; S3. Turn on the control mechanism to control the volume of the containing chamber to change, and inject a driving fluid into the containing chamber through the fluid passage to drive the second liquid to move, wherein the droplets are formed in the droplet generating tube and then flow out from the second port and enter the droplet receiver.

23. The method according to claim 22, characterized in that The first liquid is a continuous phase, and the second liquid is a dispersed phase.

24. The method according to claim 22, characterized in that The first liquid is an oil phase, and the second liquid is an aqueous phase.

25. The method according to claim 22, characterized in that A surfactant is added to the first liquid.

26. The method according to claim 22, characterized in that The second liquid is an aqueous phase containing biological or chemical substances to be detected.

27. The method according to claim 22, characterized in that The control mechanism is a vibration mechanism. In step S3, the frequency of the vibration mechanism is set to 150 Hz-300 Hz.

28. Use of the droplet generation device according to any one of claims 1 to 20, the droplet generation system according to claim 21, and the method according to any one of claims 22 to 27 in gene expression analysis or microbial detection.

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