A control device for micro-droplet preparation and a method for preparing micro-droplets

Abstract

The present invention discloses a control device for micro-droplet preparation. The control device includes a micro-droplet preparation unit. Among them, the micro-droplet preparation unit includes an asymmetric vibration micro-droplet generation mechanism and a first dynamic positioning component, and the first dynamic positioning component is fixedly connected to the asymmetric vibration micro-droplet generation mechanism; the first dynamic positioning component is configured to accurately position the asymmetric vibration micro-droplet generation mechanism; the asymmetric vibration micro-droplet generation mechanism is configured to generate micro-droplets by an asymmetric reciprocating motion mode. By using the control device of the present invention, the preparation of micro-droplets with uniform size and controllable volume can be achieved only by adjusting the flow rate of the liquid sample and the vibration frequency of the sampling needle.

Description

Technical Field

[0001] The present invention relates to the technical field of micro-droplet preparation, and in particular, to a control device for micro-droplet preparation and a method for preparing micro-droplets using the control device. Background Art

[0002] Micro-droplets are widely used in various fields. The microfluidic technology based on micro-droplets has been rapidly developed and applied in fields such as digital PCR, single-cell culture, single-cell genome / transcriptome sequencing, single-cell function sorting, high-throughput reaction screening, and protein crystallization.

[0003] The generation of micro-droplets is to generate emulsified micro-droplets using two immiscible phases. The micro-droplet phase is called the dispersed phase, and the phase that wraps the micro-droplets is called the continuous phase. After the micro-droplets are generated, operations such as splitting, fusing, mixing, diluting, collecting, and sorting can be performed on them. Therefore, it is very important to control the shape, size, and monodispersity of micro-droplets.

[0004] In the prior art, micro-droplet generation technologies are mainly divided into three types. One is to generate micro-droplets using a microfluidic chip, and its principle is based on the interfacial instability when the dispersed phase and the continuous phase meet in the microchannel. The inventor found that the generation of micro-droplets on this microfluidic chip requires specific conditions such as flow rate, oil-water interfacial tension, channel configuration, and channel surface modification. The range of micro-droplet volume adjustment is also restricted by the above factors. In addition, after the micro-droplets are generated in the microfluidic chip channel, specific steps and devices are required to transfer them to the storage container, and it is difficult to customize the conditions of individual micro-droplets. Operations such as positioning, extraction, and analysis of micro-droplets are relatively inconvenient.

[0005] The second is to form micro-droplets by spraying trace amounts of liquid using a special device, such as using special spraying or micro-droplet excitation methods such as piezoelectric ceramics, thermal expansion, and high-voltage electrospray. The inventor found that it is difficult to precisely control the volume of micro-droplets in this way, and the fluid control system matched with it is relatively complex.

[0006] The third method is that when injecting a small amount of liquid into the continuous phase through a microchannel, the outlet of the microchannel vibrates up and down at the gas-liquid interface of the continuous phase, and uniform-sized microdroplets are generated by the cutting action of the surface tension of the phase interface (Generation method of droplets based on microchannels, Du Wenbin et al., Chinese Patent No.: ZL 201410655191.5). Using this method, microdroplets with uniform size and controllable volume can be prepared. The inventors found that the microdroplet samples generated by this method need to be stored in a microinjector connected to the microchannel through a catheter. Therefore, when preparing microdroplets of multiple samples, it is necessary to replace the microchannel, catheter, and microinjector, increasing the complexity of the operation; at the same time, it is difficult for this fluid control system to prepare microdroplets for trace volume samples. In addition, when generating droplets, the microchannel needs to be accurately positioned on the liquid surface of the second liquid. The inherent error in positioning and the dynamic changes in the liquid surface during the droplet generation process will cause the microchannel to deviate or gradually deviate from the optimal positioning height, resulting in the inability to stably and continuously generate uniform droplets. Summary of the Invention

[0007] A main object of the present invention is to overcome at least one defect of the above-mentioned prior art, and to provide a control device for microdroplet preparation. The control device includes a microdroplet preparation unit, and the microdroplet preparation unit includes an asymmetric vibration microdroplet generation mechanism and a first dynamic positioning component; the first dynamic positioning component is fixedly connected to the asymmetric vibration microdroplet generation mechanism. Under the precise dynamic positioning condition of the first dynamic positioning component, the asymmetric vibration microdroplet generation mechanism drives the sampling needle to generate microdroplets in a reciprocating motion manner under the liquid surface of the second liquid, thereby avoiding the secondary emulsification and fragmentation of droplets caused by the increased disturbance of the microchannel to the oil phase due to the dynamic change of the liquid surface and the liquid addition error, and effectively ensuring the stability and consistency of the droplet generation system.

[0008] A main object of the present invention is to overcome at least one defect of the above-mentioned prior art, and to provide a control device filled with a carrier liquid in a liquid suction catheter, a liquid supply catheter, an electric two-way three-way switching valve (such as an electromagnetic two-way three-way valve or a motor-driven rotary three-way valve, etc.), a sampling needle adapter, and a sampling needle. Compared with a micropipette based on the air piston principle, the control device provided by the present invention can accurately aspirate and discharge a nanoliter volume of the first liquid sample by using the positive displacement principle; at the same time, the volume of the first liquid sample aspirated by the control device is smaller than the volume of the cavity connected to the sampling needle. Therefore, when preparing microdroplets of different first liquid samples, only the sampling needle needs to be replaced according to the microdroplet preparation method, reducing the complexity of the experimental operation.

[0009] One main object of the present invention is to overcome at least one defect of the above-mentioned prior art, and to provide a control device provided with a second open container containing a second liquid. The micro-droplets prepared by it settle and spread on the bottom of the flat sample pool in the second open container. The micro-droplets with a volume of nanoliters are protected by the second liquid, avoiding the evaporation of the micro-droplets. At the same time, the steps of transferring and storing the micro-droplets and the subsequent steps of imaging detection and analysis of the micro-droplets are simplified.

[0010] Another main object of the present invention is to overcome at least one defect of the above-mentioned prior art, and to provide a method for preparing micro-droplets using the control device. By only adjusting the flow rate of the first liquid sample and the vibration frequency of the sampling needle, the preparation of micro-droplets with uniform size and controllable volume can be achieved.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] 1. A control device for micro-droplet preparation, wherein the control device includes a micro-droplet preparation unit;

[0013] Wherein, the micro-droplet preparation unit includes an asymmetric vibration micro-droplet generation mechanism and a first dynamic positioning component, and the first dynamic positioning component is fixedly connected to the asymmetric vibration micro-droplet generation mechanism;

[0014] The first dynamic positioning component is configured to position the asymmetric vibration micro-droplet generation mechanism;

[0015] The asymmetric vibration micro-droplet generation mechanism is configured to generate micro-droplets by an asymmetric reciprocating motion mode.

[0016] 2. The control device according to item 1, wherein one micro-droplet is generated within one movement cycle of the asymmetric vibration micro-droplet generation mechanism.

[0017] 3. The control device according to item 1, wherein the asymmetric vibration micro-droplet generation mechanism includes a vibration component, a vibration mounting seat, a sampling needle, and a drive controller, wherein,

[0018] The drive controller is electrically connected to the vibration component and drives the vibration mounting seat to perform asymmetric reciprocating vibration or asymmetric reciprocating swing so that the sampling needle generates micro-droplets.

[0019] 4. The control device according to item 3, wherein within one cycle of the asymmetric reciprocating vibration or asymmetric reciprocating swing of the vibration mounting seat, one micro-droplet is generated by the sampling needle.

[0020] 5. The control device according to item 3, wherein,

[0021] The vibration assembly includes a housing, an oscillator, and a vibration output rod;

[0022] The vibration output rod of the vibration assembly provides power for the vibration mounting base;

[0023] The vibration mounting base is provided with a connection interface, a pipe joint, and a sampling needle adapter;

[0024] One end of the connection interface is connected to the liquid supply conduit through the pipe joint, and the other end is connected to the sampling needle through the sampling needle adapter;

[0025] The central axis of the sampling needle is perpendicular to the axis of the vibration output rod.

[0026] 6. The control device according to item 5, wherein the asymmetric reciprocating motion is an asymmetric reciprocating vibration of the vibration mounting base in the direction along the central axis of the vibration output rod.

[0027] 7. The control device according to item 5, wherein the asymmetric reciprocating motion is an asymmetric swing of the vibration mounting base with the central axis of the vibration output rod as the axis.

[0028] 8. The control device according to item 3 or 5, wherein the vibration frequency of the vibration assembly is 10 - 1000 Hz, preferably 50 - 200 Hz;

[0029] Preferably, the vibration amplitude of the vibration assembly is 0.1 - 5 mm, preferably 0.5 - 2 mm.

[0030] 9. The control device according to item 5, wherein the swing frequency of the vibration mounting base is 10 - 1000 Hz, preferably 50 - 200 Hz;

[0031] Preferably, the distance between the liquid discharge opening of the sampling needle and the axis of the vibration output rod is 10 - 100 mm, preferably 30 - 80 mm; the swing angle amplitude of the vibration mounting base is 0.05 - 10°, preferably 0.2 - 2°.

[0032] 10. The control device according to item 5, wherein the vibration mounting base is connected to the vibration output rod through a coupling.

[0033] 11. The control device according to item 3 or 5, wherein the vibration assembly further includes a position sensor, and the drive controller realizes closed-loop control of the motion by collecting the real-time position feedback signal of the position sensor;

[0034] Preferably, the position sensor is one of a grating scale sensor, a capacitive position sensor, a resistive sensor, a current sensor, or a differential transformer type sensor.

[0035] 12. The control device according to item 5, wherein the asymmetric vibration micro-droplet generating mechanism further includes a support fixing seat for fixing the vibration assembly;

[0036] Preferably, the asymmetric vibration micro-droplet generating mechanism further includes a pump tube clamp seat for clamping the liquid supply conduit.

[0037] 13. The control device according to item 3 or 5, wherein there are a plurality of the connection interfaces, and the plurality of connection interfaces are arranged at equal intervals inside the vibration mounting seat;

[0038] Preferably, the number of the connection interfaces is 1-96, and preferably 2, 4, 8 or 12.

[0039] 14. The control device according to item 5, wherein the asymmetric vibration micro-droplet generating mechanism further includes a connection guiding structure member, wherein,

[0040] The vibration output rod of the vibration assembly is connected to the vibration mounting seat through the connection guiding structure member to provide power for the vibration mounting seat.

[0041] 15. The control device according to item 14, wherein the connection guiding structure member is a ball spline including a spline shaft and a spline sleeve, and two ends of the spline shaft are respectively fixedly connected to the vibration output rod and the vibration mounting seat.

[0042] 16. The control device according to item 14, wherein the connection guiding structure member includes a first bearing and a second bearing, one end of the vibration mounting seat passes through the first bearing and is connected to the vibration output rod, and the other end of the vibration mounting seat is connected to the second bearing, wherein the first bearing is a bearing with an axial retaining edge.

[0043] 17. The control device according to item 3 or 5, wherein the control device further includes:

[0044] A sampling needle unloading mechanism for automatically unloading the sampling needle after micro-droplet generation.

[0045] 18. The control device according to item 5, wherein the sampling needle is a tapered tube structure with openings at both ends, one opening is a liquid supply opening for tightly plugging with the sampling needle adapter; the other opening is a liquid discharging opening for micro-droplet generation, and the inner diameter of the liquid discharging opening is 20-300 μm, and the outer diameter is 150-600 μm;

[0046] Preferably, the liquid storage volume range of the sampling needle is 5-500 μL, and preferably 20-60 μL.

[0047] 19. The control device according to item 3 or 5, wherein when the vibration mount performs the asymmetric reciprocating motion, the motion of the liquid discharging part of the sampling needle is configured to have an equilibrium point and two reflection points at both ends of the equilibrium point, and the curve of the motion position versus time is asymmetric on both sides of any reflection point;

[0048] Preferably, the asymmetric waveform of the periodic motion of the liquid discharging part of the sampling needle is an asymmetric combination of at least one of a sine wave, a sawtooth wave, a trapezoidal wave, a triangular wave, and a square wave.

[0049] 20. The control device according to item 3 or 5, wherein the vibration assembly is configured as a mechanism that generates continuous or intermittent motion, and the vibration assembly is selected from an electromagnetic vibration device, a piezoelectric ceramic vibration device, an eccentric wheel vibration device, a servo motor, a voice coil motor, and a galvanometer motor.

[0050] 21. The control device according to item 1, wherein the first dynamic positioning assembly includes:

[0051] A positioning assembly lifting displacement mechanism for controlling the lifting of the asymmetric vibration micro-droplet generating mechanism;

[0052] Preferably, the first dynamic positioning assembly further includes a liquid level detection mechanism.

[0053] 22. The control device according to item 1, wherein the control device further includes a fluid control unit,

[0054] The fluid control unit includes a fluid driving device and a conduit, one end of the conduit is connected to the fluid driving device, and the other end of the conduit is connected to the asymmetric vibration micro-droplet generating mechanism;

[0055] Preferably, the fluid driving device is used to set the flow rate to enable the sampling needle to suck and discharge liquid.

[0056] 23. The control device according to item 22, wherein the control device further includes a second positioning assembly for fixing and moving the first open container and the second open container;

[0057] Preferably, the drive controller provides power for the fluid driving device, the first dynamic positioning assembly, and the second positioning assembly.

[0058] 24. The control device according to item 23, wherein the first open container is a single liquid storage tank, a one-dimensional liquid storage tank array, or a two-dimensional liquid storage tank array, and the volume of each liquid storage tank is 10 - 1000 μL, preferably 20 - 200 μL;

[0059] Preferably, the first open container contains a first liquid;

[0060] Preferably, the second open container is a two-dimensional flat-bottom sample pool array for generating micro-droplets by tiling; preferably, the second open container includes 24, 32, 96 or 384 flat-bottom sample pools with equal volumes.

[0061] Preferably, the second open container contains a second liquid.

[0062] 25. The control device according to item 22, wherein the fluid driving device is a non-pulsating driving pump, preferably an injection pump, and further preferably, the fluid driving device is one or more.

[0063] 26. The control device according to item 22, wherein the catheter includes a liquid supply catheter and a liquid suction catheter. One end of the liquid suction catheter is connected to the fluid driving device through a valve port of an electric two-way three-way reversing valve, and the other end of the liquid suction catheter is inserted into an oil storage device; one end of the liquid supply catheter is connected to the fluid driving device through a valve port of an electric two-way three-way reversing valve, and the other end of the liquid supply catheter is connected to the asymmetric vibration micro-droplet generating mechanism.

[0064] 27. The control device according to item 1, wherein the control device further includes a preparation unit, and the preparation unit includes a sampling needle rack, an oil removal mechanism and a waste receiver. The oil removal mechanism is located above the waste receiver, and the sampling needle is unloaded above the waste receiver.

[0065] 28. A method for preparing micro-droplets using the control device according to any one of items 1-27, comprising:

[0066] Using a fluid driving device to drive a catheter, a sampling needle adapter and a sampling needle to fill the sampling needle with a first liquid;

[0067] Bringing the sampling needle filled with the first liquid into contact with the second liquid. Under the drive of the fluid driving device, the asymmetric vibration micro-droplet generating mechanism drives the sampling needle adapter to drive the sampling needle to perform an asymmetric reciprocating motion under the liquid surface of the second liquid, thereby generating micro-droplets.

[0068] 29. The method according to item 28, wherein before using the fluid driving device to drive the catheter, the sampling needle adapter and the sampling needle to fill the sampling needle with the first liquid, the method further includes:

[0069] a) Moving the sampling needle adapter to the oil removal mechanism. The fluid driving device drives the liquid suction catheter to suck the carrier liquid, switches the electric two-way three-way reversing valve, and the fluid driving device discharges the carrier liquid from the sampling needle adapter, so that the liquid suction catheter, the liquid supply catheter, the electric two-way three-way reversing valve and the sampling needle adapter are filled with the carrier liquid and there are no bubbles. At the same time, the excess carrier liquid discharged from the lower opening of the sampling needle adapter is removed by the oil removal mechanism.

[0070] b) Insert the sampling needle adapter filled with the loading liquid into the sampling needle to connect the sampling needle and the sampling needle adapter in an inserted manner;

[0071] c) Move the sampling needle to the degreasing mechanism, switch the electric two-way three-way directional valve, and repeat step a) to fill the liquid suction conduit, the liquid supply conduit, the electric two-way three-way directional valve, the sampling needle adapter, and the sampling needle with the loading liquid and without air bubbles. At the same time, the degreasing mechanism removes the excess loading liquid discharged from the opening of the sampling needle.

[0072] 30. The method according to item 29, wherein the step of using a fluid driving device to drive the conduit, the sampling needle adapter, and the sampling needle to fill the sampling needle with the first liquid includes:

[0073] Move the sampling needle in step c) above the liquid level of the first open container containing the first liquid and move downward so that the liquid outlet of the sampling needle contacts and immerses in the first liquid. Switch the electric two-way three-way directional valve to suck the first liquid into the sampling needle so that the sampling needle is filled with the first liquid.

[0074] 31. The method according to item 30, wherein the volume of the first liquid sucked from the liquid outlet of the sampling needle is less than the volume of the connection cavity of the sampling needle.

[0075] 32. The method according to item 28, wherein the method further includes:

[0076] After completing the preparation of the micro-droplets of the first liquid, unload the sampling needle, and then reinstall the sampling needle to prepare the micro-droplets of another first liquid.

[0077] 33. The method according to item 28, wherein the sampling needle performs asymmetric reciprocating vibration or asymmetric swinging under the liquid level of the second liquid, and the fluid driving device is used to set the flow rate to discharge the first liquid from the sampling needle, thereby generating micro-droplets.

[0078] 34. The method according to item 28, wherein only one micro-droplet is generated within one cycle of the asymmetric reciprocating movement of the sampling needle under the liquid level of the second liquid.

[0079] 35. The method according to item 28, characterized in that the first dynamic positioning assembly drives the asymmetric vibration micro-droplet generating mechanism to accurately position the sampling needle under the liquid level of the second liquid. During the asymmetric vibration of the sampling needle, the average depth inserted into the liquid level is dynamically maintained within the range of 0 - 2.0 mm under the liquid level, preferably within the range of 0 - 1.5 mm under the liquid level.

[0080] As can be seen from the above technical solutions, the advantages and positive effects of the micro-droplet horizontal vibration generating mechanism and the micro-droplet generating method provided by the present invention are as follows:

[0081] 1. Compared with the micropipette based on the air piston principle, for the control device provided by the present invention, the liquid suction conduit, the liquid supply conduit, the electric two-way three-way changeover valve, the sampling needle adapter and the sampling needle are filled with the carrier liquid, and the positive displacement principle can be used to accurately suck and discharge the liquid sample with a volume of nanoliters. At the same time, the volume of the liquid sample sucked by using the control device is smaller than the volume of the cavity connected to the sampling needle, so when preparing microdroplets of different samples, only the sampling needle needs to be replaced according to the microdroplet preparation method, reducing the complexity of the experimental operation.

[0082] 2. The control device provided by the present invention is provided with a control device of a second open container containing a second liquid. The microdroplets prepared by using it settle and spread on the bottom of the flat sample pool in the second open container. The nanoliter-volume microdroplets are protected by the oily second liquid, avoiding the evaporation of the microdroplets. At the same time, the steps of transferring and storing the microdroplets, as well as the subsequent steps of imaging detection and analysis of the microdroplets, are simplified.

[0083] 3. For the control device provided by the present invention, the vibration assembly of the microdroplet generating mechanism drives the liquid discharging opening of the sampling needle on the vibration mounting seat to perform a periodic reciprocating motion with an acceleration change between two points under the oil phase liquid surface or between two points across the oil phase liquid surface. By only adjusting the flow rate of the aqueous liquid in the sampling needle and the vibration frequency of the sampling needle, microdroplets with uniform size and controllable volume can be prepared.

[0084] 4. The first dynamic positioning assembly provided by the present invention is fixedly connected to the asymmetric vibration microdroplet generating mechanism. Under the precise dynamic positioning condition of the first dynamic positioning assembly, the asymmetric vibration microdroplet generating mechanism drives the sampling needle to generate microdroplets in an asymmetric reciprocating motion manner under the second liquid surface, thereby avoiding the secondary shear fragmentation of the droplets caused by the inability of the microchannel to contact the second liquid surface or the excessive insertion depth into the liquid surface due to dynamic changes in the liquid surface, liquid addition errors, sampling needle length errors, system assembly errors, etc., which may cause severe oil phase disturbances, effectively ensuring the stability, continuity and consistency of the droplet generation system.

[0085] 5. For the control device provided by the present invention, the microdroplet generating mechanism is provided with a connection guiding structural member that resists torque and radial forces, avoiding the torque and radial forces exerted on the vibration output rod by the inertial forces and gravitational forces of multiple sampling needles and the vibration mounting seat during the arrayed generation of microdroplets and the automatic loading of the sampling needle, ensuring the precision of the asymmetric reciprocating motion and the stability and uniformity of the microdroplet generation.

[0086] 6. The control device provided by the present invention, wherein the micro-droplet generation mechanism adopts an asymmetric vibration mode, ensuring that there is a unique maximum velocity position or maximum cutting force position within one cycle. The asymmetric processing defects of the sampling needle will not affect the generation of droplets, so it has strong compatibility with consumable processing defects, ensuring the reliability and uniformity of droplet preparation.

[0087] 7. The control device provided by the present invention, wherein the drive controller realizes closed-loop feedback control by means of a pre-set asymmetric vibration control program and the real-time position feedback signal of the position sensor, realizes the precise asymmetric reciprocating motion of the vibration mounting seat, and realizes the generation of highly precise and uniform nano-liter droplets.

[0088] 8. Using the micro-droplet preparation method provided by the present invention and the above-mentioned control device, by only adjusting the flow rate of the liquid sample and the vibration frequency of the sampling needle, the stable and continuous preparation of micro-droplets with uniform size and controllable volume can be realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] By considering the following detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings, various objectives, features and advantages of the present invention will become more obvious. The drawings are only exemplary illustrations of the present invention and are not necessarily drawn to scale. In the drawings, the same reference numerals always represent the same or similar components. Among them:

[0090] Figure 1 is a schematic structural diagram of the control device for preparing micro-droplets provided by the present invention.

[0091] Figure 2 is a three-dimensional structural diagram of the micro-droplet generation mechanism that makes asymmetric reciprocating vibration along the central axis direction of the vibration output rod, which is Figure 1 an enlarged view of the component labeled 1 in

[0092] Figure 3 is a sectional structural diagram of the micro-droplet generation mechanism that makes asymmetric reciprocating vibration along the central axis direction of the vibration output rod, which is Figure 1 an enlarged view of the component labeled 1 in

[0093] Figure 4 is a three-dimensional structural diagram of the micro-droplet generation mechanism that makes asymmetric swinging, which is Figure 1 an enlarged view of the component labeled 1 in

[0094] Figure 5 is a front view of the micro-droplet generation mechanism that makes asymmetric swinging, which is Figure 1 an enlarged view of the component labeled 1 in

[0095] Figure 6Schematic cross-sectional view of the micro-droplet generation mechanism for asymmetric oscillation provided by the present invention, which is Figure 1 An enlarged view of No. 1 in

[0096] Figure 7 Structure and control logic diagram of the micro-droplet generation mechanism for asymmetric oscillation provided by the present invention.

[0097] Figure 8 Configuration of the liquid ejection part of the sampling needle adapted to the micro-droplet generation mechanism provided by the present invention, (a) 3D stereoscopic view of the sampling needle; (b) cross-sectional view of the sampling needle; (c) ideal structure with a flat liquid ejection part and uniform wall thickness of the sampling needle; (d) structure with a flat liquid ejection part and uneven wall thickness of the sampling needle, resulting in left-right asymmetry of the sampling needle; (e) burr machining defect at the outer side of the liquid ejection opening of the liquid ejection part of the sampling needle; (f) burr machining defect at the inner side of the liquid ejection opening of the liquid ejection part of the sampling needle.

[0098] Figure 9 Curve of combined asymmetric vibration of sine wave and sawtooth wave adopted by the micro-droplet generation mechanism in a specific embodiment provided by the present invention. (a) Vibration position-time curve; (b) Vibration velocity-time curve.

[0099] Figure 10 Curve of combined asymmetric vibration of asymmetric sawtooth waves adopted by the micro-droplet generation mechanism in a specific embodiment provided by the present invention. (a) Vibration position-time curve; (b) Vibration velocity-time curve.

[0100] Figure 11 Curve of combined asymmetric vibration of sawtooth wave and square wave adopted by the micro-droplet generation mechanism in a specific embodiment provided by the present invention. (a) Vibration position-time curve; (b) Vibration velocity-time curve.

[0101] Figure 12 Symmetric vibration position-time curves adopted by the comparative example of the present invention. (a) Sine wave; (b) Square wave.

[0102] Figure 13 Result diagram of the micro-droplet generation mechanism generating micro-droplets in Example 1 of the present invention.

[0103] Figure 14 Result diagram of the micro-droplet generation mechanism generating micro-droplets in Example 2 of the present invention.

[0104] Figure 15 Result diagram of the micro-droplet generation mechanism generating micro-droplets with a symmetric vibration waveform in Comparative Example 1 of the present invention.

[0105] Figure 16In Comparative Example 2 of the present invention, it is a result diagram of a micro-droplet generating mechanism generating micro-droplets using a symmetric vibration waveform.

[0106] Figure 17 It is a schematic diagram of the vibration position of the micro-droplet generating mechanism that makes asymmetric reciprocating vibrations provided by the present invention.

[0107] Figure 18 It is a schematic diagram of the swinging position of the micro-droplet generating mechanism that makes asymmetric swinging provided by the present invention.

[0108] Figure 19 It is a schematic diagram of the vibration position of the micro-droplet generating mechanism that makes asymmetric reciprocating vibrations provided by the present invention.

[0109] Figure 20 It is a schematic diagram of the vibration position of the micro-droplet generating mechanism that makes asymmetric swinging provided by the present invention.

[0110] Figure 21 It is a schematic diagram of the structure of the control device for preparing micro-droplets provided by the present invention.

[0111] Figure 22 It is a schematic diagram of the generated droplets in Example 3.

[0112] Figure 23 It is a schematic diagram of the generated droplets in Example 4.

[0113] Figure 24 It is a schematic diagram of the generated droplets in Example 5.

[0114] Figure 25 It is a schematic diagram of the generated droplets in Example 6.

[0115] Figure 26 It is a schematic diagram of the droplets generated at different flow rates of the first liquid sample in Examples 7 - 18 spreading on the bottom of the container.

[0116] Figure 27 It is a schematic diagram of the relationship between the volume of the generated droplets and the theoretical micro-droplet volume in Examples 19 - 28.

[0117] Figure 28 It is a schematic diagram of different volumes of droplets generated at different concentrations of human genome samples in Example 29.

[0118] Figure 29 It is a schematic diagram of the relationship between the digital PCR quantification value and the nucleic acid sample concentration after amplification using PCR in Example 29.

[0119] Among them, 1 - asymmetric vibration micro-droplet generation mechanism, 100 - vibration component, 101 - housing, 102 - oscillator, 103 - position sensor, 104 - vibration output rod, 105 - connection guiding structure member, 1051 - spline sleeve, 1052 - spline shaft, 1053 - first bearing, 1054 - second bearing, 106 - connecting member, 120 - support fixing seat, 200 - first dynamic positioning component, 201 - positioning component lifting displacement mechanism, 202 - liquid level detection mechanism, 300 - vibration mounting seat, 301 - sampling needle adapter, 302 - pipe joint, 303 - pump tube clamp seat, 304 - connection interface, 400 - sampling needle, 401 - sampling needle liquid supply opening, 402 - sampling needle liquid discharging part, 403 - sampling needle liquid discharging opening, 404 - liquid storage cavity, 500 - fluid driving device, 501 - liquid supply conduit, 502 - liquid suction conduit, 503 - micro-syringe, 504 - oil storage bottle, 505 - carrier liquid, 600 - driving controller, 601 - vibration driving circuit, 602 - asymmetric vibration control program, 603 - position correction module, 604 - position signal acquisition module, 605 - power supply control connection cable, 700 - second liquid, 701 - second open container, 702 - second liquid level, 703 - first liquid micro-droplet, 800 - first liquid, 801 - first open container, EP - equilibrium position of vibration, RP1 and RP2 - reflection points, 900 - second positioning component. Detailed implementation manners

[0120] Typical embodiments embodying the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various variations in different embodiments, all of which do not depart from the scope of the present invention, and the descriptions and drawings therein are for illustrative purposes in essence and not for limiting the present invention.

[0121] In the following description of different exemplary embodiments of the present invention, reference is made to the accompanying drawings, which form a part of the present invention and in which different exemplary structures, systems, and steps for implementing various aspects of the present invention are shown by way of example. It should be understood that other specific solutions of components, structures, exemplary devices, systems, and steps can be used and structural and functional modifications can be made without departing from the scope of the present invention. Moreover, although terms such as "upper end portion", "lower end portion", "between", "side", etc. may be used in this specification to describe different exemplary features and elements of the present invention, these terms are used herein only for convenience, for example, according to the directions of the examples described in the drawings. Nothing in this specification should be construed as requiring a specific three-dimensional direction of the structure to fall within the scope of the present invention.

[0122] Figure 1 It is a schematic structural diagram of a control device for micro-droplet preparation disclosed by the present invention. Figure 2-7For Figure 1 An enlarged view of the asymmetric vibration micro-droplet generating mechanism 1 in Figure 1 In

[0123] The first dynamic positioning component 200 is configured to position the asymmetric vibration micro-droplet generating mechanism 1;

[0124] The asymmetric vibration micro-droplet generating mechanism 1 is configured to generate micro-droplets by an asymmetric reciprocating motion mode;

[0125] The control device further includes a fluid control unit, and the fluid control unit includes a fluid driving device 500 and a conduit (not shown in the figure). One end of the conduit is connected to the fluid driving device 500, and the other end of the conduit is connected to the asymmetric vibration micro-droplet generating mechanism 1;

[0126] The fluid driving device 500 is used to set the flow rate to enable the sampling needle 400 to aspirate and discharge liquid.

[0127] The conduit includes a liquid supply conduit 501 and a liquid suction conduit 502. One end of the liquid suction conduit 502 is connected to the fluid driving device 500 through a valve port of an electric two-way three-way switching valve (not shown in the figure), and the other end of the liquid suction conduit 502 is inserted into an oil storage device (such as an oil storage bottle 504); One end of the liquid supply conduit 501 is connected to the fluid driving device 500 through a valve port of the electric two-way three-way switching valve, and the other end of the liquid supply conduit 501 is connected to the asymmetric vibration micro-droplet generating mechanism 1.

[0128] The oil storage bottle 504 is filled with a carrier liquid 505, and the fluid driving device 500 is used to fill the liquid suction conduit 502, the liquid supply conduit 501, the electric two-way three-way switching valve, the sampling needle adapter, and the sampling needle 400 with the carrier liquid 505.

[0129] The carrier liquid can be, for example, one or more of liquid alkanes, liquid esters, and fluorinated alkanes.

[0130] In one embodiment, within one movement cycle of the asymmetric vibration micro-droplet generating mechanism, only one micro-droplet is generated.

[0131] In one embodiment, the asymmetric reciprocating motion mode of the asymmetric vibration micro-droplet generating mechanism may include asymmetric reciprocating vibration and asymmetric reciprocating swing. Within one cycle of the vibration mounting base performing asymmetric reciprocating vibration or asymmetric reciprocating swing, the sampling needle generates only one micro-droplet.

[0132] As shown Figure 2-Figure 7 in the figure, the asymmetric vibration micro-droplet generation mechanism 1 includes a vibration assembly 100, a connection and guiding structure member 105, a vibration mounting base 300, a sampling needle 400, and a drive controller 600. Among them,

[0133] the vibration assembly 100 includes a housing 101, an oscillator 102, and a vibration output rod 104;

[0134] the vibration output rod 104 of the vibration assembly 100 is connected to the vibration mounting base 300 through the connection and guiding structure member 105 to provide power for the vibration mounting base 300;

[0135] the vibration mounting base 300 is provided with a connection interface 304, a pipe joint 302, and a sampling needle adapter 301;

[0136] one end of the connection interface 304 is connected to the liquid supply conduit 501 through the pipe joint 302, and the other end is connected to the sampling needle 400 through the sampling needle adapter 301;

[0137] the central axis of the sampling needle 400 is perpendicular to the axis of the vibration output rod 104;

[0138] the drive controller 600 is electrically connected to the vibration assembly 100 and drives the vibration mounting base 300 to perform an asymmetric reciprocating motion according to the asymmetric vibration control program 602, so that the sampling needle produces micro-droplets.

[0139] In one embodiment, the drive controller 600 includes a vibration drive circuit 601, an asymmetric vibration control program 602, a position correction module 603, a position signal acquisition module 604, and a power supply control connection cable 605. The drive controller 600 is electrically connected to the vibration assembly 100 and drives the vibration mounting base 300 to perform an asymmetric reciprocating motion according to the asymmetric vibration control program 602.

[0140] In one embodiment, the asymmetric reciprocating motion is an asymmetric reciprocating vibration of the vibration mounting base 300 in the direction of the central axis of the vibration output rod 104.

[0141] In one embodiment, the connection and guiding structure member 105 is a ball spline including a spline shaft 1052 and a spline sleeve 1051. The two ends of the spline shaft 1052 are respectively fixedly connected to the vibration output rod 104 and the vibration mounting base 300. The connection between the spline shaft 1052 and the vibration output rod 104 is completed through a connecting member 106. In one embodiment, the connecting member 106 is a screw.

[0142] In one embodiment, the vibration assembly 100 provides power for the vibration mounting base 300 to perform an asymmetric reciprocating motion. It is a vibration assembly capable of generating continuous or intermittent vibrations, and is selected from one of an electromagnetic vibration device, a piezoelectric ceramic vibration device, an eccentric wheel vibration device, a servo motor, a voice coil motor, and a galvanometer motor. In the preparation of micro-droplets, the vibration frequency and vibration amplitude of the specific vibration device used can be selected according to actual needs. For example, the vibration frequency of the vibration assembly 100 is 10 - 1000 Hz, preferably 50 - 200 Hz, and the vibration amplitude is 0.1 - 5 mm, preferably 0.5 - 2 mm.

[0143] The connection and guiding structural member 105 (ball spline) can restrict the movement of the vibration mounting base 300, such that the vibration mounting base 300 can only perform a reciprocating motion in the central axis direction of the ball spline. Specifically, in the actual application of the micro-droplet generating mechanism of the present invention, when the vibration output rod 104 bears the radial force perpendicular to its axial direction generated by the weights of the vibration mounting base 300 and the sampling needle 400 that bear its load, as well as the torque force generated by the unbalanced weights at both ends of the vibration mounting base 300, vibrations other than the direction of the central axis of the vibration output rod 104 or the ball spline will not occur, such that the vibration mounting base 300 can only perform a reciprocating motion in the central axis direction of the ball spline, thereby ensuring the stability and uniformity of micro-droplet generation.

[0144] The other end of the connection and guiding structural member 105 is connected to the vibration mounting base 300. Specifically, one end of the spline shaft 1052 is connected to the vibration mounting base 300.

[0145] In one embodiment, the vibration assembly 100 further includes a position sensor 103. The drive controller 600 collects the real-time position feedback signal of the position sensor 103 of the vibration assembly 100 through the position signal acquisition module 604, compares it with the asymmetric reciprocating motion control program in real time and feeds it back to the position correction module 603 to adjust the control parameters of the vibration drive circuit 601, thereby realizing the closed-loop control of the asymmetric reciprocating motion.

[0146] The vibration assembly 100 of the present invention performs a periodic asymmetric reciprocating motion, having an equilibrium point EP (equilibrium point) and two reflecting points (reflecting-point) at both ends of the equilibrium point: RP1 and RP2. The so-called asymmetric reciprocating motion defines a vibration period as starting from the reflecting point RP1, passing through the equilibrium point EP to reach the reflecting point RP2, and then returning from the reflecting point RP2 through the equilibrium point EP to reach the reflecting point RP1. Figure 17Schematic diagram of the vibration position of the micro-droplet generation mechanism that makes asymmetric reciprocating vibrations provided by the present invention, where EP is located at the center position of RP1 and RP2, that is, the distances from EP to RP1 and RP2 are equal. Figure 18 Schematic diagram of the vibration position of the micro-droplet generation mechanism that makes asymmetric swinging motions provided by the present invention, where EP is located at the center position of RP1 and RP2, that is, the distances from EP to RP1 and RP2 are equal. Figure 19 Schematic diagram of the vibration position of the micro-droplet generation mechanism that makes asymmetric reciprocating vibrations provided by the present invention, where the distances from EP to RP1 and RP2 are not equal. Figure 20 Schematic diagram of the vibration position of the micro-droplet generation mechanism that makes asymmetric swinging motions provided by the present invention, where the distances from EP to RP1 and RP2 are not equal.

[0147] In one embodiment, for the asymmetric reciprocating motion vibration assembly of the present invention, EP is located at the center position of RP1 and RP2. Taking the position at the balance point EP as 0 for the time horizontal axis and the vibration position as the vertical axis to make a curve, the waveform curve of the vibration position versus time is asymmetric on both sides of any reflection point (RP1 or RP2) ( Figure 9 , Figure 10 and Figure 11 ). This periodic asymmetric reciprocating motion enables the liquid discharge opening of the sampling needle to reach the maximum motion speed at only one time point or one time period within the two half-cycles of the motion. The shear forces acting on the liquid discharge opening by the oil phase are different. That is, during the periodic motion of the liquid discharge opening, there is only one maximum shear force acting point within one cycle. In one embodiment, for the asymmetric reciprocating motion vibration assembly of the present invention, the distances from EP to RP1 and RP2 are not equal. The periodic asymmetric reciprocating motion enables the liquid discharge opening of the sampling needle to reach the maximum motion speed at only one time point or one time period within the two half-cycles of the motion ( Figure 19 , Figure 20 ) That is, during the periodic motion of the liquid discharge opening, there is only one maximum shear force acting interval within one cycle. Compared with the asymmetric reciprocating motion of the present invention, liquid droplets can also be generated by the left-right symmetric reciprocating vibration under the liquid surface. However, the vibration assembly has two maximum force points in one full cycle. That is, it is possible to generate liquid droplets in both half-cycles of the vibration assembly. That is, it is possible to generate a small liquid droplet in one half-cycle or a large liquid droplet with twice the volume in one full cycle. Therefore, there is a large uncertainty in the size of the liquid droplets, and the openings of the micro-channels processed by injection molding, etc. actually cannot be made completely symmetric (such as Figure 8 d, Figure 8 e, Figure 8As shown in Fig. f, there are also differences in the sizes of the generated droplets in the two half-cycles. Therefore, the present invention solves the problem of non-uniform droplet generation in the aforementioned symmetric reciprocating vibration by means of asymmetric reciprocating vibration, as well as the excessive requirements for the symmetry, precision, and defects of the processing of the liquid ejection opening, and can achieve highly controllable and uniform droplet generation.

[0148] The asymmetric reciprocating motion can take various forms. In one embodiment, the asymmetric reciprocating motion is an asymmetric reciprocating vibration along the central axis direction of the vibration output rod, that is, the vibration assembly 100 can only perform reciprocating vibration along the central axis direction of the vibration output rod 104. In one embodiment, the asymmetric reciprocating motion is an asymmetric swing centered on the central axis of the vibration output rod 104. Figure 2 and Figure 3 Fig. shows a schematic diagram of a micro-droplet generation mechanism performing asymmetric reciprocating vibration along the central axis direction of the vibration output rod.

[0149] In one embodiment, the asymmetric reciprocating motion is an asymmetric swing of the vibration mounting base centered on the central axis of the vibration output rod, as shown in Figure 4-Figure 7 Fig., which is a schematic diagram of a micro-droplet generation mechanism with an asymmetric swing centered on the central axis of the vibration output rod 104. The vibration assembly 100 includes a housing 101, an oscillator 102, a position sensor 103, and a vibration output rod 104. The vibration output rod 104 of the vibration assembly 100 is connected to the vibration mounting base 300 through a connecting guide structure member 105 and a connecting member 106, providing the power for reciprocating motion of the vibration mounting base 300. The central axis of the sampling needle 400 is perpendicular to the axis of the vibration output rod 104.

[0150] The connecting guide structure member 105 can restrict the movement of the vibration mounting base 300, so that the vibration mounting base 300 fixed on the vibration output rod 104 can only perform an asymmetric swing centered on the central axis of the vibration output rod 104. The connecting guide structure member 105 includes a first bearing 1053 and a second bearing 1054. One end of the vibration mounting base 300 passes through the first bearing 1053 and is connected to the connecting guide structure member 105, and the other end of the vibration mounting base 300 is connected to the second bearing 1054, wherein the first bearing 1053 is a bearing with an axial stop edge. In one embodiment, the connecting member 106 is a coupling. The first bearing 1053 with an axial stop edge prevents the vibration mounting base 300 from displacing in the axial direction of the vibration output rod 104, and the combined use of the first bearing 1053 and the second bearing 1054 ensures the structural stability of the vibration assembly and the stability and uniformity of micro-droplet generation.

[0151] The vibration assembly 100 provides power for the asymmetric reciprocating motion of the vibration mounting base 300 and is a mechanism capable of generating continuous or intermittent motion, which is selected from an electromagnetic vibration device, a piezoelectric ceramic vibration device, an eccentric wheel vibration device, a servo motor, a voice coil motor, and a galvanometer motor. In the preparation of micro-droplets, the vibration frequency and vibration amplitude, or the swing amplitude and swing of the specific vibration device used can be selected according to actual needs. In one embodiment, the vibration frequency is 10 - 1000 Hz, preferably 50 - 200 Hz. In one embodiment, the vibration amplitude of the vibration device is 0.1 - 5 mm, preferably 0.5 - 2 mm. In one embodiment, the swing frequency is 10 - 1000 Hz, preferably 50 - 200 Hz. In one embodiment, the distance between the liquid discharge opening of the sampling needle and the axis of the vibration output rod 104 is 10 - 100 mm, preferably 30 - 80 mm; the swing angle amplitude of the vibration mounting base is 0.05 - 10°, preferably 0.2 - 2°.

[0152] The vibration mounting base 300 of the present invention is a fixing device for the sampling needle 400 and transmits the driving force of the vibration assembly 100 to the liquid loaded in the sampling needle 400, thereby realizing the generation of micro-droplets. Among them, the central axis of the sampling needle 400 is perpendicular to the axis of the vibration output rod 104.

[0153] Specifically, the vibration mounting base 300 is provided with a connection interface 304. One end of the connection interface 304 is connected to the liquid supply conduit 501 through a pipe joint 302, and the other end is connected to the sampling needle 400 through a sampling needle adapter 301.

[0154] The connection interface 304 is a through groove and serves to connect the liquid supply conduit 501 and the sampling needle 400. According to actual application needs, the connection interface 304 is one or more, such as 1 - 96. A plurality of the connection interfaces 304 are arranged at equal intervals inside the vibration mounting base 300. Preferably, the connection interface 304 is 2, 4, 8, or 12.

[0155] Such as Figure 8 a and Figure 8As shown in Fig. b, the sampling needle 400 is a tapered tube structure with openings at both ends. One opening is the sampling needle liquid supply opening 401, which is used to tightly plug into the sampling needle adapter 301, so that the sampling needle 400 is hermetically connected to the sampling needle adapter 301. The other opening is the sampling needle liquid discharge part 402, which includes a sampling needle liquid discharge opening 403 for micro-droplet generation. The inner diameter of the sampling needle liquid discharge opening 403 is 20 - 300 μm, and the outer diameter is 150 - 600 μm. The middle of the sampling needle is a liquid storage cavity 404 for storing the first liquid 800. The volume range of the liquid storage cavity 404 of the sampling needle 400 is 5 - 500 μL, preferably 20 - 60 μL. Figure 8 Figs. c - f show the configurations of the liquid discharge part 402 of the sampling needle 400 adapted to the asymmetric reciprocating motion mechanism for micro-droplet generation provided by the present invention. Among them, Figure 8 Fig. c shows the configuration of the liquid discharge part without burrs and symmetric left and right in the ideal case after machining. By using the asymmetric reciprocating vibration of the micro-droplet generation mechanism of the present invention, the defects of the configuration of the liquid discharge part 402 of the sampling needle can be tolerated, including left - right asymmetry ( Figure 8 Fig. d), the presence of burrs on the outer side of the liquid discharge opening ( Figure 8 Fig. e), and the presence of burrs on the inner side of the liquid discharge opening ( Figure 8 Fig. f). The sizes of the processing defects are in the order of ten to one hundred micrometers. Under the existing processing conditions, they cannot be completely avoided, which has an adverse effect on the uniformity of droplet preparation.

[0156] As Figure 9-Figure 11 shown, the position of the liquid discharge opening of the sampling needle 400 swings left and right around the vibration center position. The curve of the position of the sampling needle liquid discharge opening 403 changing with time can present various forms, such as a sine wave, a sawtooth wave, a trapezoidal wave, a triangular wave, a square wave, or a superposition combination of the above multiple waveforms. In one embodiment, the curve of the position of the liquid discharge opening of the sampling needle 400 changing with time is a combination of a sine wave and a sawtooth wave (as shown in Figure 9 Fig. a), and its periodic velocity - time curve is as shown in Figure 9 Fig. b; In one embodiment, the curve of the position of the liquid discharge opening of the sampling needle 400 changing with time is a combination of a short - period sawtooth wave and a long - period sawtooth wave, as shown in Figure 10 Fig. a, and its periodic velocity - time curve is as shown in Figure 10 Fig. b; In one embodiment, the curve of the position of the liquid discharge opening of the sampling needle 400 changing with time is a combination of a square wave and a sawtooth wave (as shown in Figure 11 Fig. a), and its periodic velocity - time curve is as shown in Figure 11 Fig. b.

[0157] The characteristics of the above waveform are that the velocity change curves of the liquid discharge opening of the sample addition needle 400 in the half stroke from left to right and the half stroke from right to left are asymmetric, and in one vibration cycle, there is a maximum instantaneous velocity or a maximum velocity range greater than the average velocity. Under the action of the asymmetric reciprocating motion, the liquid discharge part of the sample addition needle 400 generates a liquid droplet within one reciprocating vibration period, and the generation of the liquid droplet is controlled by the maximum instantaneous velocity or the maximum velocity range within the reciprocating vibration cycle, so it has extremely high stability and reliability.

[0158] In one embodiment, the vibration generating mechanism of the present invention further includes a support fixing seat 120 for fixing the vibration assembly 100 to realize operations such as automatic loading, moving, and liquid droplet preparation of the sample addition needle.

[0159] In one embodiment, the vibration generating mechanism of the present invention further includes a pump tube clamp seat 303 (see Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 ) for clamping the liquid supply conduit 501. Specifically, during use, the pump tube clamp seat 303 can keep the liquid supply conduit 501 vertical, thereby ensuring stable liquid supply and making the generated micro liquid droplets stable and uniform.

[0160] In one embodiment, the first dynamic positioning assembly 200 includes a positioning assembly lifting displacement mechanism 201 for controlling the lifting and precise positioning of the asymmetric vibration micro liquid droplet generating mechanism 1.

[0161] The first dynamic positioning assembly 200 further includes a liquid level detection mechanism 202 to assist the first dynamic positioning assembly 200 in accurately positioning the opening of the sample addition needle to a fixed depth below the second liquid level 702 in the second opening container 701, and making dynamic height position adjustments under the control of the control device when the liquid level height changes due to the continuous generation of liquid droplets or there is a liquid level height error caused by the volume error of the second liquid addition, so as to ensure the long-term stability and uniformity of liquid droplet generation.

[0162] The liquid level detection mechanism 202 can be, for example, a high-definition CCD camera, a reflective photoelectric sensor, or a capacitive distance sensor.

[0163] In one embodiment, the control device further includes a second positioning assembly 900, which is a precision two-axis horizontal translation stage for fixing and moving the first opening container and the second opening container.

[0164] In one embodiment, in addition to driving and controlling the asymmetric vibration micro-droplet generating mechanism 1, the driving controller 600 simultaneously drives and controls the fluid driving device 500, the first dynamic positioning assembly 200, and the second positioning assembly 900 to work in cooperation with the asymmetric vibration micro-droplet generating mechanism 1.

[0165] In one embodiment, the first open container 801 ( Figure 1 , Figure 21 ) contains a first liquid 800.

[0166] The first liquid can be, for example, a PCR (Polymerase Chain Reaction) reagent, an RT-PCR (Reverse Transcription PCR) reagent, a microbial suspension, or a cell suspension, etc.

[0167] The first open container is a single reservoir, a one-dimensional reservoir array, or a two-dimensional reservoir array, preferably an eight-strip PCR tube, more preferably a standard 96- or 384-well microplate or PCR plate. The bottom of the reservoir can be flat-bottomed, round-bottomed, or conical-bottomed, and its volume is 10 - 1000 μL, preferably 20 - 200 μL.

[0168] For example, the volume of each first open container can be 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1000 μL, etc.

[0169] As Figure 1 and Figure 21 shown, a second liquid 700 is pre-loaded in the second open container 701, and the specific gravity of the second liquid is less than that of the first liquid; the second open container is a two-dimensional flat-bottomed sample pool array for spreading the generated micro-droplets; preferably, the second open container includes 24, 32, 96, or 384 flat-bottomed sample pools with equal volumes, and the shape of the flat-bottomed sample pool is rectangular or circular.

[0170] The second liquid is one or more of liquid alkanes or liquid esters containing surfactants, and the surfactants are one or more of Triton series surfactants, Tween series surfactants, Nonidet series surfactants, Pluronic series surfactants, Span series surfactants, Brij series surfactants, IGEPAL series surfactants, and ABIL EM / WE series surfactants.

[0171] The Triton series surfactants may be, for example, Triton X-100, Triton X-405, Triton X-114, etc.

[0172] The Tween series surfactants may be, for example, Tween 40, Tween 60, Tween 65, Tween 80, Tween 85, etc.

[0173] The Nonidet series surfactants may be, for example, P40.

[0174] The Pluronic series surfactants may be, for example, Pluronic F-68.

[0175] The Span series surfactants may be, for example, Span 20, Span 40, Span 60, Span 80, Span 85, etc.

[0176] The Brij series surfactants may be, for example, Brij30, Brij35, Brij58, Brij97, etc.

[0177] The IGEPAL series surfactants may be, for example, IGEPAL CO-630, IGEPAL CO-730, IGEPAL CO-520, IGEPAL CO-897, etc.

[0178] The ABIL EM / WE series surfactants may be, for example, ABIL EM 97, ABIL WE 09, etc.

[0179] In one embodiment, the sampling needle adapter is a tapered tube structure with one end open, and the sampling adapter is used for plugging the sampling needle;

[0180] Preferably, the sampling needle adapter includes a liquid supply conduit connection part, a connection cavity, and a liquid outlet from top to bottom;

[0181] Preferably, the inner diameter of the connection cavity is 0.5 - 1.5 mm, preferably 1 mm;

[0182] The outer diameter of the connection cavity is 2 - 4 mm, preferably 2 - 3 mm;

[0183] The length of the connection cavity is 10 - 50 mm, preferably 15 - 25 mm.

[0184] In one embodiment, the number of the sampling needle adapters is 1 - 96, preferably 2, 4, 8, or 12.

[0185] In one embodiment, the fluid driving device is a non-pulsating driving pump, preferably an injection pump.

[0186] The number of the fluid driving devices is one or more.

[0187] In one embodiment, the liquid supply conduit 501, the liquid suction conduit 502, the electric two-position three-way reversing three-way valve, and the sampling needle adapter 301 are filled with the carrier liquid 505 and have no air bubbles.

[0188] In one embodiment, the control device further includes a preparation unit (not shown in the figure), the preparation unit includes a sampling needle holder, an oil removal mechanism, a sampling needle unloading mechanism, and a waste receiver. The oil removal mechanism is located above the waste receiver, and the sampling needle is unloaded above the waste receiver.

[0189] For the sampling needle holder, the oil removal mechanism, the sampling needle unloading mechanism, and the waste receiver, the present invention does not impose any restrictions, and those skilled in the art can select according to needs. For example, the oil removal mechanism can be a sponge.

[0190] The present invention provides a method for preparing microdroplets using the above-described control device, which may include the following steps:

[0191] Using the fluid driving device to drive the conduit, the sampling needle adapter, and the sampling needle to be filled with the first liquid;

[0192] Place the opening of the sampling needle filled with the first liquid below the liquid level of the second liquid and keep it in contact with the second liquid. Under the drive of the fluid driving device, the microdroplet generating mechanism drives the sampling needle adapter to drive the sampling needle to perform an asymmetric reciprocating motion below the liquid level of the second liquid, thereby generating microdroplets of the first liquid.

[0193] In one embodiment, the sampling needle performs an asymmetric reciprocating vibration or an asymmetric swing below the liquid level of the second liquid, and the fluid driving device is used to set the flow rate to discharge the first liquid from the sampling needle, thereby generating microdroplets.

[0194] In one embodiment, within one cycle of the asymmetric reciprocating motion of the sampling needle below the liquid level of the second liquid, only one microdroplet is generated.

[0195] In one embodiment, before using the fluid driving device to drive the conduit, the sampling needle adapter, and the sampling needle to fill the sampling needle with the first liquid, the method may further include one or more of the following steps:

[0196] a) Move the sampling needle adapter to the oil removal mechanism. The fluid driving device drives the liquid suction conduit to suck the carrier liquid, switch the electric two-position three-way reversing valve, and the fluid driving device discharges the carrier liquid from the sampling needle adapter, so that the liquid suction conduit, the liquid supply conduit, the electric two-position three-way reversing valve, and the sampling needle adapter are filled with the carrier liquid and have no air bubbles. At the same time, the excess carrier liquid discharged from the lower opening of the sampling needle adapter is removed by the oil removal mechanism;

[0197] b) Insert the sampling needle adapter filled with the carrier liquid into the sampling needle to connect the sampling needle and the sampling needle adapter in a plug-in manner;

[0198] c) Move the sampling needle to the degreasing mechanism, switch the electric two-way three-way directional valve, and repeat step a) to fill the liquid suction conduit, liquid supply conduit, electric two-way three-way directional valve, sampling needle adapter, and sampling needle with the carrier liquid and without air bubbles. At the same time, the degreasing mechanism removes the excess carrier liquid discharged from the opening of the sampling needle.

[0199] In one embodiment, the step of using a fluid driving device to drive the conduit, sampling needle adapter, and sampling needle to fill the sampling needle with the first liquid may include:

[0200] Move the sampling needle in step c) above the liquid level of the first open container containing the first liquid and move downward so that the liquid outlet of the sampling needle contacts and immerses in the first liquid. Switch the electric two-way three-way directional valve to suck the first liquid into the sampling needle so that the sampling needle is filled with the first liquid.

[0201] In one embodiment, the method may include:

[0202] a) Move the sampling needle adapter to the degreasing mechanism. The fluid driving device drives the liquid suction conduit to suck the carrier liquid, switch the electric two-way three-way directional valve, and the fluid driving device discharges the carrier liquid from the sampling needle adapter to fill the liquid suction conduit, liquid supply conduit, electric two-way three-way directional valve, and sampling needle adapter with the carrier liquid and without air bubbles. At the same time, the degreasing mechanism removes the excess carrier liquid discharged from the lower opening of the sampling needle adapter;

[0203] b) Insert the sampling needle adapter filled with the carrier liquid into the sampling needle to connect the sampling needle and the sampling needle adapter in a plug-in manner;

[0204] c) Move the sampling needle to the degreasing mechanism, switch the electric two-way three-way directional valve, and repeat step a) to fill the liquid suction conduit, liquid supply conduit, electric two-way three-way directional valve, sampling needle adapter, and sampling needle with the carrier liquid and without air bubbles. At the same time, the degreasing mechanism removes the excess carrier liquid discharged from the opening of the sampling needle;

[0205] d) Move the sampling needle in step c) above the liquid level of the first open container containing the sample and move downward so that the liquid outlet of the sampling needle contacts and immerses in the first liquid. Switch the electric two-way three-way directional valve and repeat step a) to suck the first liquid into the sampling needle;

[0206] e) Move the sampling needle in step d) away from the first open container and move it above the second open container containing the second liquid, and move downward so that the liquid outlet of the sampling needle contacts and immerses in the second liquid;

[0207] f) The first liquid in the sampling needle is discharged from the liquid outlet of the sampling needle driven by the fluid driving device. At the same time, the micro-droplet generating mechanism drives the sampling needle adapter to drive the sampling needle to vibrate asymmetrically or swing asymmetrically under the liquid surface of the second liquid, so that the discharged first liquid detaches from the liquid outlet of the sampling needle to generate micro-droplets.

[0208] In one embodiment, the volume of the sample inhaled from the liquid outlet of the sampling needle is less than the volume of the connecting cavity of the sampling needle to prevent the inhaled first liquid from contaminating the liquid supply cannula.

[0209] In one embodiment, the method further includes: after completing the preparation of the micro-droplets of the first liquid, unloading the sampling needle, and then inserting another sampling needle according to steps b)-f) to perform the preparation of the micro-droplets of another first liquid.

[0210] The present invention prepares micro-droplets by using the above-mentioned device. Since the liquid discharge opening of the sampling needle on the mounting seat driven by the vibration component in the micro-droplet production mechanism performs a periodic reciprocating motion with an acceleration change between two points under the oil-phase liquid surface or across two points of the oil-phase liquid surface, by only adjusting the flow rate of the aqueous-phase liquid in the sampling needle and the vibration frequency of the sampling needle, the preparation of micro-droplets with uniform size and controllable volume is achieved.

[0211] Example 1

[0212] As Figure 1 shown, the control device includes a micro-droplet preparation unit. The micro-droplet preparation unit includes an asymmetric vibration micro-droplet generating mechanism 1 and a first dynamic positioning component 200. The first dynamic positioning component 200 is fixedly connected to the asymmetric vibration micro-droplet generating mechanism 1; the first dynamic positioning component 200 is configured to position the asymmetric vibration micro-droplet generating mechanism 1;

[0213] The asymmetric vibration micro-droplet generating mechanism 1 is configured to generate micro-droplets by an asymmetric reciprocating motion mode;

[0214] The control device may further include a fluid control unit (not shown in the figure). The fluid control unit includes a fluid driving device 500, a liquid supply conduit 501, a liquid suction conduit 502, and an electric two-way three-way reversing valve. One valve port of the electric two-way three-way reversing valve is connected to the fluid driving device 500, the second valve port is connected to one end of the liquid supply conduit 501, the third valve port is connected to one end of the liquid suction conduit 502. The other end of the liquid suction conduit 502 is connected to an oil storage bottle 504, and the other end of the liquid supply conduit 501 is connected to the asymmetric vibration micro-droplet generating mechanism 1.

[0215] The first dynamic positioning component 200 includes a positioning component lifting and displacement mechanism 201, and the asymmetric vibrating micro-droplet generating mechanism 1 is fixedly connected to the positioning component lifting and displacement mechanism 201 of the first dynamic positioning component 200.

[0216] The first dynamic positioning component 200 may further include a liquid level detection mechanism 202 to assist the first dynamic positioning component in accurately positioning the opening of the sampling needle below the second liquid level 702 in the second open container 701 ( Figure 21 ) to a depth of 0.3 mm.

[0217] The control device further includes a second positioning component 900, which is used to fix and move the first open container 801 and the second open container 701.

[0218] The first open container 801 is a single liquid storage tank, and the first open container 801 contains a first liquid;

[0219] The second open container 701 is a two-dimensional flat-bottomed sample pool array for generating micro-droplets in a tiled manner;

[0220] The control device may further include a preparation unit (not shown in the figure), the preparation unit includes a sampling needle holder, an oil removal mechanism (sponge), a sampling needle unloading mechanism, and a waste receiver. The oil removal mechanism (sponge) is located above the waste receiver, and the sampling needle is unloaded above the waste receiver.

[0221] Among them, the micro-droplet generating mechanism is as Figure 7 shown, and it includes a vibration component 100, a connection and guiding structure member 105, a vibration mounting seat 300, a sampling needle 400, and a drive controller 600.

[0222] As Figure 2 and Figure 3 shown, the micro-droplet generating mechanism in this embodiment performs asymmetric reciprocating vibration along the central axis direction of the vibration output rod. The micro-droplet generating mechanism includes a vibration component, a connection and guiding structure member 105, a support and fixing seat 120, a vibration mounting seat 300, a sampling needle 400, and a pump tube clamp seat 303. The vibration component 100 includes a housing 101, an oscillator 102, a position sensor 103, and a vibration output rod 104. One end of the vibration output rod 104 is connected to the housing 101, and the other end is connected to one end of the connection and guiding structure member 105 through a connecting member 106. The other end of the connection and guiding structure member 105 is connected to the vibration mounting seat 300. The central axis of the sampling needle 400 is perpendicular to the axis of the vibration output rod 104. The support and fixing seat 120 is used to fix the vibration component 100.

[0223] Among them, the connection guiding structural member 105 is a ball spline including a spline shaft 1052 and a spline sleeve 1051. Both ends of the spline shaft 1052 are fixedly connected to the vibration output rod 104 and the vibration mounting base 300 respectively. The connection between the spline shaft 1052 and the vibration output rod 104 is completed through a connecting member 106. The connecting member 106 is a screw. Part of the position sensor 103 is connected to the vibrator and moves with the vibrator or the vibration output rod, and the other part is fixed on the spline sleeve 1051, which can precisely detect the displacement during the vibration process.

[0224] The vibration assembly 100 is a vibration motor. The vibration frequency of the vibrator 102 is 100 - 200 Hz, and the vibration amplitude range is 0.1 - 5 mm. In actual application, the amplitude range is 0.5 - 1.5 mm.

[0225] The vibration mounting base 300 is provided with a connection interface 304. One end of the connection interface 304 is connected to the liquid supply conduit 501 through a pipe joint 302, and the other end is connected to the sampling needle 400 through a sampling needle adapter 301. There are 8 connection interfaces 304, and the 8 connection interfaces 304 are arranged at equal intervals inside the vibration mounting base 300. The pump tube clamp seat 303 is used to clamp the liquid supply conduit 501 so that the liquid supply conduit 501 remains vertical.

[0226] The drive controller 600 includes a vibration drive circuit 601, an asymmetric vibration control program 602, a position correction module 603, a position signal acquisition module 604, and a power supply control connection cable 605. The drive controller 600 is electrically connected to the vibration assembly 100 and drives the vibration mounting base 300 to perform asymmetric reciprocating motion according to the asymmetric vibration control program 602. The drive controller 600 collects the real-time position feedback signal of the position sensor 103 of the vibration assembly 100 through the position signal acquisition module 604, and feeds it back to the position correction module 603 by comparing it with the asymmetric reciprocating vibration control program in real time, and adjusts the control parameters of the vibration drive circuit 601 to achieve the closed-loop control of the asymmetric reciprocating motion.

[0227] The drive controller 600 is also electrically connected to the first dynamic positioning assembly 200, the second positioning assembly 900, and the fluid drive device 500 to provide power for them respectively. When the liquid level changes due to the continuous generation of liquid droplets or there is a liquid level height error caused by the volume error of the second liquid addition, the drive controller 600 controls the first dynamic positioning assembly 200 to make dynamic height position adjustments to ensure the long-term stability and uniformity of liquid droplet generation.

[0228] The method for preparing micro-droplets using the above-mentioned control device may include the following steps:

[0229] a) Move the sample addition needle adapter to the sponge. The injection pump drives the liquid suction catheter to suck the carrier liquid. Switch the electric two-way three-way directional valve. The injection pump discharges the carrier liquid from the sample addition needle adapter, so that the liquid suction catheter, the liquid supply catheter, the electric two-way three-way directional valve, and the sample addition needle adapter are filled with the carrier liquid and there are no air bubbles. At the same time, the sponge removes the excess carrier liquid discharged from the lower opening of the sample addition needle adapter. The above carrier liquid is a second liquid that is immiscible with the sample liquid. The carrier liquid is an oil mainly composed of hydrocarbons, silicon, or fluorocarbons, such as mineral oil, tetradecane, FC40, etc.;

[0230] b) Insert the sample addition needle adapter filled with the carrier liquid into the sample addition needle to make the sample addition needle and the sample addition needle adapter connected by insertion. Among them, the liquid storage volume of the sample addition needle is 60 μL. The material for preparing the sample addition needle 400 is polypropylene (PP, the contact angle with pure aqueous solution is 88°). The inner diameter of the liquid discharge opening of the sample addition needle 400 is 120 μm, and the outer diameter is 400 μm;

[0231] c) Move the sample addition needle to the sponge, switch the electric two-way three-way directional valve, and repeat step a) to make the liquid suction catheter, the liquid supply catheter, the electric two-way three-way directional valve, the sample addition needle adapter, and the sample addition needle filled with the carrier liquid and there are no air bubbles. At the same time, the sponge removes the excess carrier liquid discharged from the opening of the sample addition needle;

[0232] d) Move the sample addition needle in step c) above the liquid level of the first open container containing the first liquid and move downward so that the liquid outlet of the sample addition needle touches and immerses in the aqueous solution (the first liquid). Switch the electric two-way three-way directional valve and repeat step a) to suck 20 μL of the first liquid into the sample addition needle;

[0233] e) Move the sample addition needle in step d) away from the first open container and move it above the second open container containing mineral oil (the second liquid). The first dynamic component drives the asymmetric vibration micro-droplet generation mechanism to move downward so that the liquid outlet of the sample addition needle touches and immerses 0.3 mm below the liquid level of the mineral oil;

[0234] f) The first liquid in the sample addition needle is discharged from the liquid outlet of the sample addition needle under the drive of the injection pump. The drive controller 600 is used to realize the drive control of the asymmetric periodic reciprocating movement of the sample addition needle under or across the liquid level of the second liquid. The vibration motor in the micro-droplet generation mechanism drives the sample addition needle adapter to drive the liquid discharge opening of the sample addition needle to make an asymmetric reciprocating movement. The position-time waveform of the movement is a combination of a sine wave and a sawtooth wave, as Figure 9 shown. The vibration amplitude is 1.2 mm, the vibration frequency is 100 Hz, the flow rate of the microinjector is 100 nL / s, and the injection volume is 20 μL. Using the above parameter conditions, 1 nL micro-droplets are prepared. The microscopic imaging of the micro-droplets is as Figure 13 shown. The CV of the droplet volume size is 1.8%.

[0235] Example 2

[0236] As Figure 1 shown, the control device includes a micro-droplet preparation unit, the micro-droplet preparation unit includes an asymmetric vibration micro-droplet generation mechanism 1 and a first dynamic positioning component 200, and the first dynamic positioning component 200 is fixedly connected to the asymmetric vibration micro-droplet generation mechanism 1; the first dynamic positioning component 200 is configured to position the asymmetric vibration micro-droplet generation mechanism 1;

[0237] The asymmetric vibration micro-droplet generation mechanism 1 is configured to generate micro-droplets by an asymmetric reciprocating motion mode;

[0238] The control device further includes a fluid control unit (not shown in the figure), the fluid control unit includes a fluid driving device 500, a liquid supply conduit 501, a liquid suction conduit 502 and an electric two-way three-way changeover valve; one valve port of the electric two-way three-way changeover valve is connected to the fluid driving device 500, the second valve port is connected to one end of the liquid supply conduit 501, the third valve port is connected to one end of the liquid suction conduit 502, the other end of the liquid suction conduit 502 is connected to an oil storage bottle 504, and the other end of the liquid supply conduit 501 is connected to the asymmetric vibration micro-droplet generation mechanism 1.

[0239] The first dynamic positioning component 200 includes a positioning component lifting displacement mechanism 201, and the asymmetric vibration micro-droplet generation mechanism 1 is fixedly connected to the positioning component lifting displacement mechanism 201 of the first dynamic positioning component 200.

[0240] As Figure 21 The first dynamic positioning component 200 further includes a liquid level detection mechanism 202 to assist the first dynamic positioning component to accurately position the opening of the sampling needle below the second liquid level 702 in the second open container 701 by 0.3 mm. The liquid level detection and sensing mechanism is a high-definition CCD camera, which performs real-time imaging on the process of the sampling needle inserting into the liquid level, and judges the distance between the sampling needle and the liquid level and the insertion state through image analysis. The above high-definition CCD camera assists the first dynamic positioning component to accurately position the opening of the sampling needle to a fixed depth below the second liquid level in the second open container, and when the liquid level changes due to the continuous generation of droplets or there is a liquid level height error caused by the volume error of the second liquid addition, it assists the first dynamic positioning component to make dynamic height position adjustments under the control of the control device to ensure the long-term stability and uniformity of droplet generation.

[0241] The control device further includes a second positioning component 900, and the second positioning component is used to fix and move the first open container 801 and the second open container 701.

[0242] The first open container 801 is a single liquid storage tank, and a first liquid 800 is contained in the first open container 801;

[0243] The second open container 701 is a two-dimensional flat-bottomed sample cell array for generating microdroplets by spreading;

[0244] The control device further includes a preparation unit (not shown in the figure). The preparation unit includes a sampling needle holder, an oil removal mechanism (sponge), a sampling needle unloading mechanism, and a waste receiver. The oil removal mechanism (sponge) is located above the waste receiver, and the sampling needle is unloaded above the waste receiver.

[0245] Among them, the microdroplet generation mechanism is as Figure 7 shown, and it includes a vibration assembly 100, a connection and guiding structure member 105, a vibration mounting seat 300, a sampling needle 400, and a drive controller 600.

[0246] As Figure 4-Figure 7 shown, the microdroplet generation mechanism in this embodiment drives the vibration mounting seat 300 to swing asymmetrically with the central axis of the vibration output rod 104 as the axis. The microdroplet generation mechanism includes a vibration assembly 100, a connection and guiding structure member 105, a support and fixing seat 120, a vibration mounting seat 300, a sampling needle 400, a support and fixing seat 120, and a pump tube clamp seat 303. The vibration assembly 100 includes a housing 101, an oscillator 102, a position sensor 103, and a vibration output rod 104. One end of the vibration output rod 104 is connected to the housing 101, and the other end is connected to the vibration mounting seat 300 through the connection and guiding structure member 105 and a connecting member 106. The central axis of the sampling needle 400 is perpendicular to the axis of the vibration output rod 104.

[0247] The connection and guiding structure member 105 includes a first bearing 1053 and a second bearing 1054. One end of the vibration mounting seat 300 passes through the first bearing 1053 and is connected to the connection and guiding structure member 105, and the other end of the vibration mounting seat 300 is connected to the second bearing 1054, where the first bearing 1053 is a bearing with an axial retaining edge. The connecting member 106 is a coupling.

[0248] The vibration assembly 100 is a vibration motor. The vibration frequency of the oscillator 102 is 100 - 500 Hz, and the vibration amplitude range is 0.1 - 5 mm. In actual application, the vibration frequency is 100 - 200 Hz, and the amplitude range is 0.5 - 1.5 mm.

[0249] The vibration mounting base 300 is provided with a connection interface 304. One end of the connection interface 304 is connected to the liquid supply conduit 501 through a pipe joint 302, and the other end is connected to the sampling needle 400 through a sampling needle adapter 301. There are 4 connection interfaces 304, and the 4 connection interfaces 304 are arranged at equal intervals inside the vibration mounting base 300. The pump tube clamp seat 303 is used to clamp the liquid supply conduit 501 to keep the liquid supply conduit 501 vertical.

[0250] The drive controller 600 includes a vibration drive circuit 601, an asymmetric vibration control program 602, a position correction module 603, a position signal acquisition module 604, and a power supply control connection cable 605. The drive controller 600 is electrically connected to the vibration assembly 100 and drives the vibration mounting base 300 to perform asymmetric reciprocating motion according to the asymmetric vibration control program 602. The drive controller 600 collects the real-time position feedback signal of the position sensor 103 of the vibration assembly 100 through the position signal acquisition module 604, and feeds it back to the position correction module 603 by comparing it with the asymmetric reciprocating vibration control program in real time, and adjusts the control parameters of the vibration drive circuit 601 to realize the closed-loop control of the asymmetric reciprocating motion.

[0251] The drive controller 600 is also electrically connected to the first dynamic positioning assembly 200, the second positioning assembly 900, and the fluid drive device 500 to provide power for them respectively.

[0252] The method for preparing micro-droplets using the above-mentioned control device includes the following steps:

[0253] a) Move the sampling needle adapter to the sponge. The syringe pump drives the liquid suction conduit to suck the carrier liquid, switch the electric two-way three-way reversing valve, and the syringe pump discharges the carrier liquid from the sampling needle adapter, so that the liquid suction conduit, the liquid supply conduit, the electric two-way three-way reversing valve, and the sampling needle adapter are filled with the carrier liquid and there are no air bubbles. At the same time, the sponge removes the excess carrier liquid discharged from the lower opening of the sampling needle adapter. The above carrier liquid is a second liquid that is immiscible with the sample liquid. The carrier liquid is an oil mainly composed of hydrocarbons, silicon, or fluorocarbons, such as mineral oil, tetradecane, FC40, etc.;

[0254] b) Insert the sampling needle adapter filled with the carrier liquid into the sampling needle to make the sampling needle and the sampling needle adapter connected by plugging. Among them, the liquid storage volume of the sampling needle is 60 μL. The material of the prepared sampling needle 400 is polypropylene (PP, the contact angle of the pure aqueous solution is 88°). The inner diameter of the liquid ejection opening of the sampling needle 400 is 120 μm, and the outer diameter is 400 μm;

[0255] c) Move the sampling needle to the sponge, switch the electric two-way three-way directional valve, and repeat step a) to fill the liquid suction catheter, liquid supply catheter, electric two-way three-way directional valve, sampling needle adapter, and sampling needle with the carrier liquid and without air bubbles. At the same time, remove the excess carrier liquid discharged from the opening of the sampling needle by the sponge;

[0256] d) Move the sampling needle in step c) above the liquid level of the first open container containing the first liquid, and move downward so that the liquid outlet of the sampling needle contacts and immerses in the aqueous solution (the first liquid). Switch the electric two-way three-way directional valve and repeat step a) to suck 20 μL of the first liquid into the sampling needle;

[0257] e) Move the sampling needle in step d) away from the first open container and move it above the second open container containing mineral oil (the second liquid). Move downward so that the liquid outlet of the sampling needle contacts and immerses in the second liquid, and the depth of immersion of the sampling needle into the liquid level of the second liquid is 0.3 mm;

[0258] f) The first liquid in the sampling needle is discharged from the liquid outlet of the sampling needle under the drive of the syringe pump. Use the drive controller 600 as the drive signal generator for the sampling needle to perform asymmetric periodic reciprocating motion under or across the liquid level of the second liquid. The vibration motor in the micro-droplet generation mechanism drives the sampling needle adapter to drive the liquid discharge opening of the sampling needle to perform asymmetric reciprocating motion. The position-time waveform of the motion is a combination of a square wave and a sawtooth wave, as Figure 11 shown. The vibration frequency is 100 Hz, the amplitude is 1.2 mm, the flow rate of the syringe pump is 100 nL / s, and the injection volume is 20 μL. Micro-droplets of 1 nL are prepared. The microscopic imaging of the micro-droplets is as Figure 14 shown. The volume of the liquid droplet is 1 nL, and the CV value of the volume is 2.1%.

[0259] Comparative Example 1

[0260] The difference between Comparative Example 1 and Example 1 is that the micro-droplet generation mechanism performs symmetric reciprocating vibration along the central axis direction of the vibration output rod, and the vibration waveform is a sine wave (as Figure 12 shown in a), the vibration amplitude is 1.2 mm, the vibration frequency is 100 Hz, the flow rate of the micro-syringe is 100 nL / s, and the injection volume is 20 μL. Others, including the structure of the micro-droplet generation mechanism and the conditions of the micro-droplet preparation method, are the same as those in Example 1. The prepared micro-droplets are as Figure 15 shown. The volume of the liquid droplets is not uniform, and the CV value of the volume is 35%. Further microscopic high-speed imaging observation found that since droplet cutting occurs in both half cycles of the vibration, and due to defects such as burrs in the processing of the sampling needle, in some cases, the liquid droplets at the liquid discharge opening of the sampling needle in one cycle cannot be cut off by the oil-phase shear force, resulting in the easy generation of 1 nL, 1.5 nL, or 2 nL liquid droplets.

[0261] Comparative Example 2

[0262] The difference between Comparative Example 2 and Example 1 is that the micro-droplet generation mechanism performs symmetric reciprocating vibration along the central axis direction of the vibration output rod, and the vibration waveform is a square wave (as shown in Figure 12 b), the amplitude is 1.2 mm, the vibration frequency is 200 Hz, the flow rate of the micro-syringe is 200 nL / s, and the injection volume is 20 μL. Other conditions including the structure of the micro-droplet generation mechanism and the micro-droplet preparation method are the same as those in Example 1. The prepared micro-droplets are as shown in Figure 16 . The CV value of the droplet size is 15%. Through further microscopic high-speed imaging observation, it is found that within one vibration cycle, a droplet is generated at the liquid discharge opening of the sampling needle in both the upper half cycle and the lower half cycle. However, due to defects such as the non-coaxial processing of the sampling needle, the volume sizes of the droplets generated in the upper half cycle and the lower half cycle are inconsistent, introducing a large volume error.

[0263] Comparative Example 3

[0264] The difference between Comparative Example 3 and Example 1 is that the micro-droplet generation mechanism does not include the connecting and guiding structural member 105. That is, the vibration output rod 104 is directly connected to the vibration mounting base 300 through the connecting member 106. Other structures are the same as those in Example 1. The micro-droplets are prepared by using the same micro-droplet preparation method as in Example 1. This vibration mechanism has a large vibration noise and obvious resonance; the CV value of the volume of the prepared micro-droplets is 7.5%. Compared with Example 1, the uniformity of the micro-droplet volume is poor, introducing a large volume error. When installing the sampling needle, due to the lack of the connecting and guiding structural member, the vibration assembly cannot resist the torque acting force and the radial acting force. After using it 34 times, the connection between the galvanometer vibration output rod and the vibration motor is bent, and normal sampling needle insertion cannot be achieved.

[0265] Comparative Example 4

[0266] The difference between Comparative Example 4 and Example 2 is that the micro-droplet generation mechanism swings symmetrically around the central axis of the vibration output rod 104. Other conditions including the structure of the micro-droplet generation mechanism and the micro-droplet preparation method are the same as those in Example 2. The specific parameters of the symmetric reciprocating motion are that the vibration waveform is a square wave ( Figure 12 b), the vibration amplitude is 1.2 mm, the vibration frequency is 100 Hz, the flow rate of the micro-syringe is 100 nL / s, and the injection volume is 20 μL. The uniformity of the prepared micro-droplets is poor, and the CV value of the micro-droplet volume is 8.5%, introducing a large volume error.

[0267] Comparative Example 5

[0268] The difference between Comparative Example 5 and Example 2 is that the micro-droplet generation mechanism does not include the connection guiding structure member 105. That is, the vibration output rod 104 is directly connected to the vibration mounting base 300 through the connecting member 106. Other structures are the same as those in Example 2. The micro-droplets are prepared by the same micro-droplet preparation method as in Example 2. The prepared micro-droplets are as shown in Figure 12 Figure a. The specific parameters of the symmetric reciprocating motion are that the vibration waveform is a sine wave, the vibration amplitude is 1.2 mm, the vibration frequency is 100 Hz, the flow rate of the micro-syringe is 100 nL / s, and the injection volume is 20 μL. The uniformity of the prepared micro-droplets is poor, and the CV value of the micro-droplet volume is 7.4%, introducing a large volume error. The vibration mechanism has a large vibration noise and obvious resonance; when installing the sampling needle, due to the lack of the connection guiding structure member, the vibration assembly cannot resist the torque force and the radial force. After using it 25 times, the galvanometer vibration output rod is bent and the normal installation of the sampling needle cannot be achieved.

[0269] Comparative Example 6

[0270] The difference between Comparative Example 6 and Example 2 is that the micro-droplet generation mechanism does not include the sampling needle adapter 301. That is, the liquid supply conduit 501 passes through the vibration mounting base 300 and is directly connected to the sampling needle 400. Other structures are the same as those in Example 2. The micro-droplets are prepared by the same micro-droplet preparation method as in Example 2. The prepared micro-droplets are as shown in Figure 12 Figure a. The specific parameters of the symmetric reciprocating motion are that the vibration waveform is a sine wave, the vibration amplitude is 1.2 mm, the vibration frequency is 100 Hz, the flow rate of the micro-syringe is 100 nL / s, and the injection volume is 20 μL. The uniformity of the prepared micro-droplets is poor, and the CV value of the micro-droplet volume is 30%, introducing a large volume error. When installing the sampling needle, due to the absence of the sampling needle adapter, the airtightness is poor, and it is easy to cause inaccurate droplet generation volume due to leakage.

[0271] Among them, the vibration component parameters and droplet generation results of the above-mentioned examples and comparative examples are shown in Table 1.

[0272] Table 1 Vibration component parameters and droplet generation results of each example

[0273]

[0274]

[0275] The above examples show that by using the asymmetric reciprocating motion proposed in the present invention, it is possible to effectively avoid the non-uniformity of droplet sizes caused by the processing defects of the sampling needle and the injection molding error at the opening, resulting in asymmetry.

[0276] Examples 3-6

[0277] Examples 3-6 are different from Example 1 in that the depth of the first dynamic positioning component for positioning the sampling needle is 0.5 mm, 1.0 mm, 1.5 mm, and 2.5 nm below the liquid surface. The asymmetric vibration micro-droplet generation mechanism is used to generate droplets. After spreading them flat on the bottom of the container, microscope imaging is used for observation, and the results are as follows.

[0278] Example 3: The sampling needle is positioned 0.5 mm below the liquid surface, and the generated droplets are as Figure 22 shown. The generated droplets are uniform, with a volume of 1 nL.

[0279] Example 4: The sampling needle is positioned 1.0 mm below the liquid surface, and the generated droplets are as Figure 23 shown. The generated droplets are uniform, with a volume of 1 nL.

[0280] Example 5: The sampling needle is positioned 1.5 mm below the liquid surface, and the generated droplets are as Figure 24 shown. The generated droplets are uniform, with a volume of 1 nL.

[0281] Example 6: The sampling needle is positioned 2.5 mm below the liquid surface, and the generated droplets are as Figure 25 shown. When generating, the droplets are uniform, but the droplets are partially broken due to the disturbance of the oil phase.

[0282] Experiments found that when the sampling needle is located 0.0 - 1.5 mm below the liquid surface, the generated droplets are uniform, with accurate volume. During the process of generating droplets, the droplets can settle orderly and will not be disturbed by the sampling needle.

[0283] The experimental results of Examples 3-6 show that for the control device for preparing micro-droplets proposed in the present invention, an asymmetric vibration micro-droplet generation mechanism needs to cooperate with the first dynamic positioning component to effectively control the depth of the sampling needle inserted into the liquid surface, ensuring the uniformity of droplet size and the stability and reliability of the droplet generation system. In the actual use process of the droplet generation device, an array of sampling needles is often used to generate droplets in an array of containers. The machining errors, assembly errors of multiple sampling needles, and the liquid surface errors caused by the volume error of the oil phase in the container are all inevitable; in addition, as the number of generated droplets increases, the liquid surface of the second liquid will gradually rise, gradually causing the depth of the sampling needle inserted into the liquid surface to gradually deviate from the allowable depth range. The first dynamic positioning component with dynamic height adjustment function can ensure that the above errors and the liquid surface changes during the droplet generation process will not affect the stability and reliability of the system.

[0284] Examples 7-18

[0285] Examples 7-18 are different from Example 1 in that the fixed vibration frequency is 120 Hz, and the flow rates of the first liquid sample are set to 120 nL / s, 240 nL / s, 360 nL / s, 480 nL / s, 600 nL / s, 720 nL / s, 840 nL / s, 960 nL / s, 1080 nL / s, 1200 nL / s, 1320 nL / s, and 1440 nL / s respectively for variation. Under the above conditions, the asymmetric vibration micro-droplet generation mechanism generates droplets. After the generated droplets are flattened on the bottom of the container, microscope imaging is used for observation. The results are as Figure 26 shown. It is found that the volumes of the generated droplets are 1 nL, 2 nL, 3 nL, 4 nL, 5 nL, 6 nL, 7 nL, 8 nL, 9 nL, 10 nL, 11 nL, and 12 nL respectively. The droplet sizes are uniform, and the droplet volume is equal to the flow rate divided by the vibration frequency.

[0286] Examples 7-18 verify the advantages of the micro-droplet preparation control device proposed by the present invention in controllable droplet generation.

[0287] Examples 19-28

[0288] Examples 19-28 are different from Example 1 in that the flow rate of the first liquid sample and the vibration frequency of the sampling needle are as shown in Table 2. Using the control device for micro-droplet preparation, through asymmetric vibration, the theoretical volume of the generated droplet is the flow rate divided by the vibration frequency. The generated droplet volumes in Examples 19-28 are in good agreement with the actual and theoretical volumes of the generated droplets. The linear correlation between the generated droplet volume and the theoretical droplet volume value is in good agreement with the theoretical calculation (R 2 = 0.9999, Figure 27 ) and the volumes are 200 pL, 500 pL, 1 nL, 5 nL, 10 nL, 50 nL, 100 nL, 500 nL, 1 μL, and 2 μL respectively.

[0289] Examples 19-28 further prove that by using the present invention, droplets with adjustable sizes can be quickly generated by adjusting the flow rate, amplitude, and frequency. The generation conditions are flexibly controllable, and controllable generation of micro-droplets with adjustable volumes spanning five orders of magnitude (200 pL to 2 μL) can be achieved, far exceeding the adjustable range of droplet generation by the existing microfluidic chip method, and without changing the structure of the sampling needle. Compared with the existing microfluidic chip method for droplet generation, the device provided by the present invention can directly set the droplet size, without the need to observe the droplet volume and optimize parameters according to experience to determine the droplet size. The droplet volume is not affected by the change in the inner diameter of the sampling needle and has very good consistency.

[0290] Table 2 Generation of droplets using the asymmetric vibration micro-droplet generation mechanism by adjusting the flow rate, amplitude, and frequency

[0291]

[0292]

[0293] Example 29

[0294] This example verifies the performance of multiplex volume digital PCR detection using the control device for microdroplet preparation proposed by the present invention. Conventional digital PCR instruments, such as the QX200 of Bio-Rad Laboratories, Inc. in the United States, the QuantStudio3D of Thermo Fisher Scientific in the United States, and the Naica of Stilla in France, etc., produce droplets or microreactions with a fixed volume, and the volume of the droplets is between 0.5 - 0.8 nL. The dynamic linear detection range is usually 10 5 orders of magnitude. The dynamic range is relatively narrow, and a trade-off is made between sensitivity and quantification limit. This example uses the control device for microdroplet preparation to generate a droplet array of multiple predetermined volumes as needed, further providing the accuracy and dynamic detection range of digital PCR. The upper quantification limit of microdroplets of different volumes was estimated based on the classical digital PCR Poisson distribution model. It was calculated that as the droplet volume decreases, the detection upper limit of droplet quantification will increase accordingly. This is mainly because under the same reaction system, the smaller the microdroplet, the more units can be divided, so that at the same concentration, the ratio of the number of positive droplets to the total number of droplets is different, and thus the detection limit will be improved. Then, based on the theoretical model, the quantitative dynamic range of microdroplets of different volumes was theoretically calculated to predict the dynamic range of digital PCR linear detection under different droplet volume conditions. The 95% confidence quantification intervals of 0.2 nL, 0.5 nL, 1 nL, 2.5 nL, and 5 nL volume droplets are shown as Figure 29 dotted lines of different formats. The 95% confidence quantification intervals for the five droplet volumes are shown as Figure 29 the solid curve. It can be seen that through different volume methods, the dynamic quantification range can theoretically be expanded from 10 5 orders of magnitude to 10 6 orders of magnitude (as shown in Table 3).

[0295] Table 3. Upper quantification limit, lower detection limit, and detection error of multiplex volume digital PCR

[0296]

[0297]

[0298] Table note: [1] The upper quantification limit for each condition is the concentration observed when the fraction of positive droplets is 0.95; [2] The lower detection limit for each condition is 5 positive droplets in one reaction.

[0299] The actual experimental steps are as follows:

[0300] The volume of the configured digital PCR reaction system is 25 μL, which consists of 12.5 μL of 2× dPCR Super Mix (Beijing Dawi Biotechnology Co., Ltd.), 6.9 μL of deionized water, 0.6 μL of DNA polymerase (Beijing Dawi Biotechnology Co., Ltd.), 2.5 μL of 10× PCR primer probe, and 2.5 μL of nucleic acid template. The target to be detected is the EIF5B (eukaryotic translation initiation factor 5B) gene in human genomic DNA (gDNA) (as shown in Table 4).

[0301] Table 4. Primer and probe sequences for digital PCR detection of EIF5B gene

[0302]

[0303] The gDNA sample (TaqMan Control Genomic DNA, Applied Biosystems, USA) was serially diluted in TE buffer to concentrations of 100,000, 10,000, 1,000, 100, 10, and 1 copy / μL. Pure water was used as a blank control. Under the same other experimental conditions as in Example 1, using the control device for microdroplet preparation, at a frequency of 120 Hz, flow rates of 24 nL / s, 60 nL / s, 120 nL / s, 300 nL / s, and 600 nL / s were respectively used to successfully generate droplets of 0.2 nL, 0.5 nL, 1 nL, 2.5 nL, and 5 nL. The results are as Figure 28 shown. The generated droplets formed a planar monolayer droplet array in a flat-bottomed well plate (Beijing Dawi Biotechnology Co., Ltd.). The flat-bottomed well plate was amplified on a flat-plate PCR amplifier (Beijing Dawi Biotechnology Co., Ltd.) using the following PCR program: 95°C for 5 minutes, 45 cycles of 94°C for 20 seconds and 58°C for 1 minute, and finally held at 25°C. After amplification, the flat-bottomed well plate was transferred to a fluorescence reader for droplet fluorescence image acquisition, and the results were analyzed using digital PCR analysis software to obtain the absolute quantification results of the EIF5B gene at different concentrations and different droplet volumes.

[0304] The results are as Figure 29 shown. It can be seen from Figure 29 that by generating microdroplets of different volumes such as 0.2 nL, 0.5 nL, 1 nL, 2.5 nL, and 5 nL, in the range of 1 - 10 5The dynamic range of detection and quantification was investigated within the range of the concentration of human genomic gDNA template at copies / μL. Linear regression was performed on the quantification results of each volume of droplets. The R values for the quantification of 0.2 nL, 0.5 nL, 1 nL, 2.5 nL, and 5 nL droplets were 0.979, 0.980, 0.995, 0.993, and 0.987 respectively; while the R value for the combined quantification of all volume droplets was 0.996.

[0305] The above results indicate that by comprehensively statistically analyzing the quantification results of droplets of different volumes, the dynamic range of quantitative detection of digital PCR can be greatly expanded, thereby improving the accuracy of quantification. The linear range of the current mainstream fluorescence quantitative PCR is in the range of 5 - 7 log10 concentration ranges, and the linear range of imported digital PCR based on chips is usually in the range of 4 - 5 log10 concentration ranges, which is narrower than that of fluorescence quantitative PCR. The control device for droplet generation proposed in the present invention not only greatly reduces the consumable cost of digital PCR, realizes the automated preparation of droplet arrays, but also can further expand the linear dynamic range of digital PCR through the multi - volume technology, popularize the applications of digital PCR in the quantitative detection of virus load, highly sensitive detection of rare mutations, etc., which is of great significance.

[0306] The exemplary embodiments of the control device for micro - droplet generation proposed in the present invention have been described and / or illustrated in detail above. However, the embodiments of the present invention are not limited to the specific embodiments described herein. On the contrary, each component and / or step of each embodiment can be used independently and separately from other components and / or steps described herein. Each component and / or each step of one embodiment can also be combined with other components and / or steps of other embodiments. When introducing the elements / components / etc. described and / or illustrated herein, terms such as "a", "one", and "the above" are used to indicate the existence of one or more elements / components / etc. The terms "comprising", "including", and "having" are used to mean an open - ended inclusion, which means that there may be additional elements / components / etc. in addition to the listed elements / components / etc. In addition, the terms "first" and "second" etc. in the claims and the specification are only used as labels and are not numerical limitations on their objects. Sequence Listing <110> Beijing Dawi Microbial Technology Co., Ltd. <120> A Control Device for Micro - droplet Preparation and a Method for Preparing Micro - droplets <130> TPE01695 <150> CN2021109855081 <151> 2021 - 08 - 26 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Artificially synthesized sequence <400> 1 atgagatgcc aaacttcagc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Artificially synthesized sequence <400> 2 ggcaacattt cacactacag 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Artificially synthesized sequence <400> 3 ctcttctcat gcagttgtca gaag 24

Claims

1. A control device for micro-droplet preparation, characterized in that, the control device includes a micro-droplet preparation unit; wherein, the micro-droplet preparation unit includes an asymmetric vibration micro-droplet generation mechanism and a first dynamic positioning component, and the first dynamic positioning component is fixedly connected to the asymmetric vibration micro-droplet generation mechanism; the first dynamic positioning component is configured to position the asymmetric vibration micro-droplet generation mechanism; the asymmetric vibration micro-droplet generation mechanism is configured to generate micro-droplets by an asymmetric reciprocating motion mode; one micro-droplet is generated within one motion cycle of the asymmetric vibration micro-droplet generation mechanism; the asymmetric vibration micro-droplet generation mechanism includes a vibration component, a connection guiding structure member, a vibration mounting seat, a sampling needle, and a driving controller; the vibration component includes a housing, an oscillator, and a vibration output rod, and the vibration output rod of the vibration component is connected to the vibration mounting seat through the connection guiding structure member to provide power for the vibration mounting seat; the driving controller is electrically connected to the vibration component and drives the vibration mounting seat to perform asymmetric reciprocating vibration or asymmetric reciprocating swing so that the sampling needle generates micro-droplets; the liquid discharge opening of the sampling needle has only one maximum shear force stress point within one cycle; the micro-droplet detaches from the opening when the liquid discharge opening of the sampling needle reaches the maximum shear force stress point, and the size of the micro-droplet is positively correlated with the flow rate of the first liquid sample, and the size of the micro-droplet is inversely correlated with the vibration frequency of the vibration component.

2. The control device according to claim 1, characterized in that, within one cycle of the asymmetric reciprocating vibration or asymmetric reciprocating swing of the vibration mounting seat, the sampling needle generates one micro-droplet.

3. The control device according to claim 1, characterized in that, the vibration mounting seat is provided with a connection interface, a pipe joint, and a sampling needle adapter; one end of the connection interface is connected to the liquid supply conduit through the pipe joint, and the other end is connected to the sampling needle through the sampling needle adapter; the central axis of the sampling needle is perpendicular to the axis of the vibration output rod.

4. The control device according to claim 3, characterized in that, the asymmetric reciprocating motion is the asymmetric reciprocating vibration of the vibration mounting seat along the central axis direction of the vibration output rod.

5. The control device according to claim 3, characterized in that, the asymmetric reciprocating motion is the asymmetric swing of the vibration mounting seat with the central axis of the vibration output rod as the axis.

6. The control device according to claim 1 or 3, characterized in that, the vibration frequency of the vibration component is 10 - 1000 Hz.

7. The control device according to claim 1 or 3, characterized in that, the vibration frequency of the vibration component is 50 - 200 Hz.

8. The control device according to claim 1 or 3, characterized in that, the vibration amplitude of the vibration component is 0.1 - 5 mm.

9. The control device according to claim 1 or 3, characterized in that, the vibration amplitude of the vibration component is 0.5 - 2 mm.

10. The control device according to claim 3, characterized in that, The swing frequency of the vibration mounting base is 10 - 1000 Hz.

11. The control device according to claim 3, characterized in that the swing frequency of the vibration mounting base is 50 - 200 Hz.

12. The control device according to claim 3, characterized in that the distance between the liquid discharge opening of the sample addition needle and the axis of the vibration output rod is 10 - 100 mm.

13. The control device according to claim 3, characterized in that the distance between the liquid discharge opening of the sample addition needle and the axis of the vibration output rod is 30 - 80 mm.

14. The control device according to claim 3, characterized in that the swing angle amplitude of the vibration mounting base is 0.05 - 10°.

15. The control device according to claim 3, characterized in that the swing angle amplitude of the vibration mounting base is 0.2 - 2°.

16. The control device according to claim 3, characterized in that the vibration mounting base is connected to the vibration output rod through a coupling.

17. The control device according to claim 1 or 3, characterized in that the vibration assembly further includes a position sensor, and the drive controller realizes closed-loop control of the movement by collecting the real-time position feedback signal of the position sensor.

18. The control device according to claim 17, characterized in that the position sensor is one of a grating scale sensor, a capacitive position sensor, a resistive sensor, a current sensor or a differential transformer type sensor.

19. The control device according to claim 3, characterized in that the asymmetric vibration micro-droplet generating mechanism further includes a support fixing base for fixing the vibration assembly.

20. The control device according to claim 1, characterized in that the asymmetric vibration micro-droplet generating mechanism further includes a pump tube clamping seat for clamping the liquid supply conduit.

21. The control device according to claim 1 or 3, characterized in that there are multiple connection interfaces, and the multiple connection interfaces are arranged at equal intervals inside the vibration mounting base.

22. The control device according to claim 21, characterized in that the number of connection interfaces is 1 - 96.

23. The control device according to claim 21, characterized in that the number of connection interfaces is 2, 4, 8 or 12.

24. The control device according to claim 1, characterized in that the connection guiding structure member is a ball spline including a spline shaft and a spline sleeve, and both ends of the spline shaft are fixedly connected to the vibration output rod and the vibration mounting base respectively.

25. The control device according to claim 1, characterized in that the connection guiding structure member includes a first bearing and a second bearing, one end of the vibration mounting base passes through the first bearing and is connected to the vibration output rod, and the other end of the vibration mounting base is connected to the second bearing, wherein the first bearing is a bearing with an axial retaining edge.

26. The control device according to claim 1, characterized in that the control device further includes: a sample addition needle unloading mechanism for automatically unloading the sample addition needle after micro-droplet generation.

27. The control device according to claim 1, characterized in that, the sampling needle is a tapered tubular structure with openings at both ends. One opening is a liquid supply opening for tightly plugging into the sampling needle adapter; the other opening is a liquid discharging opening for generating micro-droplets. The inner diameter of the liquid discharging opening is 20 - 300 μm, and the outer diameter is 150 - 600 μm.

28. The control device according to claim 1, characterized in that, the liquid storage volume range of the sampling needle is 5 - 500 μL.

29. The control device according to claim 1, characterized in that, the liquid storage volume range of the sampling needle is 20 - 60 μL.

30. The control device according to claim 1 or 3, characterized in that, when the vibration mounting base performs the asymmetric reciprocating motion, the motion of the liquid discharging part of the sampling needle is configured to have an equilibrium point and two reflection points at both ends of the equilibrium point, and the curve of the motion position versus time is asymmetric on both sides of any reflection point.

31. The control device according to claim 30, characterized in that, the asymmetric waveform of the periodic motion of the liquid discharging part of the sampling needle is an asymmetric combination of at least one of sine wave, sawtooth wave, trapezoidal wave, triangular wave, and square wave.

32. The control device according to claim 1 or 3, characterized in that, the vibration assembly is configured as a mechanism for generating continuous or intermittent motion, and the vibration assembly is selected from one of electromagnetic vibration devices, piezoelectric ceramic vibration devices, eccentric wheel vibration devices, servo motors, voice coil motors, and galvanometer motors.

33. The control device according to claim 1, characterized in that, the first dynamic positioning assembly includes: a positioning assembly lifting displacement mechanism for controlling the lifting of the asymmetric vibration micro-droplet generating mechanism.

34. The control device according to claim 1, characterized in that, the first dynamic positioning assembly further includes a liquid level detection mechanism.

35. The control device according to claim 1, characterized in that, the control device further includes a fluid control unit, the fluid control unit includes a fluid driving device and a conduit. One end of the conduit is connected to the fluid driving device, and the other end of the conduit is connected to the asymmetric vibration micro-droplet generating mechanism.

36. The control device according to claim 35, characterized in that, the fluid driving device is used to set the flow rate for the sampling needle to suck and discharge liquid.

37. The control device according to claim 35, characterized in that, the control device further includes a second positioning assembly for fixing and moving the first open container and the second open container.

38. The control device according to claim 37, characterized in that, the drive controller provides power for the fluid driving device, the first dynamic positioning assembly, and the second positioning assembly.

39. The control device according to claim 37, characterized in that, the first open container is a single liquid storage pool, a one-dimensional liquid storage pool array, or a two-dimensional liquid storage pool array, and the volume of each liquid storage pool is 10 - 1000 μL.

40. The control device according to claim 39, characterized in that, The volume of each liquid storage pool is 20 - 200 μL.

41. The control device according to claim 39, wherein, the first open container contains a first liquid.

42. The control device according to claim 39, wherein, the second open container is a two-dimensional flat-bottom sample pool array for generating micro-droplets by spreading.

43. The control device according to claim 42, wherein, the second open container includes 24, 32, 96 or 384 flat-bottom sample pools with equal volumes.

44. The control device according to claim 39, wherein, the second open container contains a second liquid.

45. The control device according to claim 35, wherein, the fluid driving device is a non-pulsating driving pump.

46. The control device according to claim 35, wherein, the fluid driving device is an injection pump.

47. The control device according to claim 35, wherein, the fluid driving device is one or more.

48. The control device according to claim 35, wherein, the catheter includes a liquid supply catheter and a liquid suction catheter. One end of the liquid suction catheter is connected to the fluid driving device through a valve port of an electric two-position three-way reversing valve, and the other end of the liquid suction catheter is inserted into an oil storage device; one end of the liquid supply catheter is connected to the fluid driving device through a valve port of the electric two-position three-way reversing valve, and the other end of the liquid supply catheter is connected to the asymmetric vibration micro-droplet generating mechanism.

49. The control device according to claim 1, wherein, the control device further includes a preparation unit, and the preparation unit includes a sampling needle holder, an oil removal mechanism and a waste receiver. The oil removal mechanism is located above the waste receiver, and the sampling needle is unloaded above the waste receiver.

50. A method for preparing micro-droplets using the control device according to any one of claims 1 - 49, comprising: using a fluid driving device to drive the catheter, the sampling needle adapter and the sampling needle to fill the sampling needle with the first liquid; bringing the sampling needle filled with the first liquid into contact with the second liquid, and under the drive of the fluid driving device, the asymmetric vibration micro-droplet generating mechanism drives the sampling needle adapter to drive the sampling needle to perform an asymmetric reciprocating motion under the liquid surface of the second liquid, thereby generating micro-droplets.

51. The method according to claim 50, wherein, before using the fluid driving device to drive the catheter, the sampling needle adapter and the sampling needle to fill the sampling needle with the first liquid, the method further includes: a) moving the sampling needle adapter to the oil removal mechanism, the fluid driving device drives the liquid suction catheter to suck the carrier liquid, switches the electric two-position three-way reversing valve, and the fluid driving device discharges the carrier liquid from the sampling needle adapter, so that the liquid suction catheter, the liquid supply catheter, the electric two-position three-way reversing valve and the sampling needle adapter are filled with the carrier liquid, and at the same time, the oil removal mechanism removes the excess carrier liquid discharged from the lower opening of the sampling needle adapter; b) inserting the sampling needle adapter filled with the carrier liquid into the sampling needle to make the sampling needle and the sampling needle adapter be plugged and connected; c) Move the sampling needle to the degreasing mechanism, switch the electric two-way three-way directional valve, and repeat step a) to fill the liquid suction catheter, liquid supply catheter, electric two-way three-way directional valve, sampling needle adapter, and sampling needle with the carrier liquid and without air bubbles. At the same time, the degreasing mechanism removes the excess carrier liquid discharged from the opening of the sampling needle.

52. The method according to claim 51, wherein, the step of using a fluid driving device to drive the catheter, sampling needle adapter, and sampling needle to fill the sampling needle with the first liquid includes: Move the sampling needle in step c) above the liquid level of the first open container containing the first liquid and move downward so that the liquid outlet of the sampling needle contacts and immerses in the first liquid. Switch the electric two-way three-way directional valve to suck the first liquid into the sampling needle so that the sampling needle is filled with the first liquid.

53. The method according to claim 52, wherein, the volume of the first liquid sucked from the liquid outlet of the sampling needle is less than the volume of the connection cavity of the sampling needle.

54. The method according to claim 50, wherein, the method further includes: After completing the preparation of the micro-droplets of the first liquid, unload the sampling needle, and then reinstall the sampling needle to prepare the micro-droplets of another first liquid.

55. The method according to claim 50, wherein, the sampling needle performs asymmetric reciprocating vibration or asymmetric swinging under the liquid level of the second liquid, and the fluid driving device is used to set the flow rate to discharge the first liquid from the sampling needle, thereby generating micro-droplets.

56. The method according to claim 50, wherein, only one micro-droplet is generated within one cycle of the asymmetric reciprocating movement of the sampling needle under the liquid level of the second liquid.

57. The method according to claim 50, wherein, the first dynamic positioning component drives the asymmetric vibration micro-droplet generating mechanism to position the sampling needle under the liquid level of the second liquid. During the asymmetric vibration of the sampling needle, the average depth of insertion into the liquid level is dynamically maintained within the range of 0 - 2.0 mm below the liquid level.

58. The method according to claim 50, wherein, during the asymmetric vibration of the sampling needle, the average depth of insertion into the liquid level is dynamically maintained within the range of 0 - 1.5 mm below the liquid level.

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