Control device and method for producing microdroplets

Abstract

A control device for micro-droplet production, the control device includes a micro-droplet production means, the micro-droplet production means includes an asymmetric vibration micro-droplet production mechanism (1) and a first dynamic positioning member (200), the first dynamic positioning member (200) is fixedly connected to the asymmetric vibration micro-droplet production mechanism (1), the first dynamic positioning member (200) is configured to accurately position the asymmetric vibration micro-droplet production mechanism (1), and the asymmetric vibration micro-droplet production mechanism (1) is configured to produce micro-droplets by an asymmetric reciprocating motion method. By using the control device, it is possible to realize the production of micro-droplets with uniform size and controllable volume simply by adjusting the flow rate of the liquid sample and the vibration frequency of the sample addition needle.

Description

Technical Field

[0001] This application relates to the technical field of micro-droplet generation, and specifically, to a control device for micro-droplet generation and a method for generating micro-droplets using the control device.

Background Art

[0002] Micro-droplets are widely applied in various fields, and the micro-fluid control technology based on micro-droplets has seen rapid development and application in fields such as digital PCR, single-cell culture, single-cell genomics / transcriptome sequencing, single-cell function sorting, high-throughput screening, protein crystallization, etc.

[0003] The generation of micro-droplets is to generate micro-droplets emulsified using two immiscible phases. The micro-droplet phase is called the dispersed phase, and the phase that encloses 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, the control of the shape, size, and monodispersity of micro-droplets is important.

[0004] In the prior art, the micro-droplet generation technology is mainly divided into three types. The first is to generate micro-droplets using a micro-fluidic chip, and its principle is based on the interfacial destabilization when the dispersed phase and the continuous phase merge in the micro-channel. The inventors have found that for the generation of micro-droplets in such a micro-fluidic chip, it is necessary to meet conditions such as specific flow rates, oil-water interfacial tension, channel arrangement, and channel surface modification, and the range of micro-droplet volume adjustment is also restricted by the above factors. Also, after the micro-droplets are generated in the channels of the micro-fluidic chip, it is necessary to transfer them to a storage container through specific steps and devices, and it is difficult to customize the conditions of a single micro-droplet, and operations such as positioning, extraction, and analysis of micro-droplets are inconvenient.

[0005] The second method involves using a special device to spray a small amount of liquid to form microdroplets. Special spraying or microdroplet excitation methods such as piezoelectric ceramics, thermally excited expansion, and high-pressure spraying are employed. The inventors found that precisely controlling the volume of microdroplets using such methods is difficult, and that the corresponding fluid control system is complex.

[0006] The third method involves injecting a small amount of liquid into a continuous phase via a micropipe, vibrating the outlet of the micropipe up and down at the gas-liquid interface of the continuous phase, and utilizing the cutting action of the surface tension at the phase interface to generate microdroplets of uniform size (Micropipe-based droplet generation method, Du Wenbin et al., Chinese Patent No.: ZL201410655191.5). This method makes it possible to produce microdroplets of uniform size and controllable volume. The inventors found that because the microdroplet samples produced by this method need to be stored in a microsyringe connected to the micropipe via a conduit, it is necessary to change the micropipe, conduit, and microsyringe when producing microdroplets for multiple samples, which increases the complexity of the operation, and that this fluid control system makes it difficult to produce microdroplets for minute volumes of samples. Furthermore, once droplets are generated, the micropipe needs to be precisely positioned on the surface of the second liquid. However, inherent positioning errors and dynamic changes in the liquid surface during the droplet generation process can cause the micropipe to shift or gradually deviate from its optimal positioning height, making it impossible to stably and continuously generate uniform droplets. [Overview of the project] [Means for solving the problem]

[0007] One of the main objectives of this application is to overcome at least one of the defects of the prior art described above and to provide a control device for producing microdroplets, the control device including a microdroplet production means, the microdroplet production means including an asymmetric vibrating microdroplet generation mechanism and a first dynamic positioning member, the first dynamic positioning member being fixedly connected to the asymmetric vibrating microdroplet generation mechanism, and under precise dynamic positioning conditions of the first dynamic positioning member, the asymmetric vibrating microdroplet generation mechanism drives a sample addition needle to generate microdroplets in an asymmetric reciprocating motion manner below the liquid surface of a second liquid, thereby avoiding secondary emulsification and fragmentation of droplets caused by disturbances to the oil phase of the micropipe due to dynamic changes in the liquid surface and liquid addition errors, and effectively ensuring the stability and consistency of the droplet generation system.

[0008] One of the main objectives of this application is to overcome at least one of the defects of the prior art described above and to provide a control device that fills a liquid suction conduit, a liquid supply conduit, an electric two-position three-way switching valve (e.g., an electromagnetic two-position three-way valve or a motor-driven rotary three-way valve), a sample dosing needle adapter, and a sample dosing needle with a carrier liquid. Compared to micropipettes based on the air piston principle, the control device provided by this application can accurately aspirate and discharge a first liquid sample of nanoliter volume using the positive displacement principle. At the same time, because the volume of the first liquid sample aspirated by the control device is smaller than the volume of the connection cavity of the sample dosing needle, when creating microdroplets of different first liquid samples, the sample dosing needle can be replaced according to the microdroplet preparation method, thereby reducing the complexity of experimental operations.

[0009] One of the main objectives of the present invention is to overcome at least one drawback of the prior art described above and to provide a control device equipped with a second open container containing a second liquid, thereby preventing evaporation of the microdroplets, as the microdroplets produced therein settle and are stacked flat at the bottom of a flat-bottomed sample cell in the second open container, and the nanoliter volume of microdroplets is protected by the second liquid. This also simplifies the microdroplet transfer and storage steps and the subsequent microdroplet imaging detection and analysis steps.

[0010] Another main objective of this application is to overcome at least one drawback of the above-mentioned prior art and to provide a method for producing microdroplets using the control device, which enables the production of microdroplets of uniform size and controllable volume simply by adjusting the flow rate of the first liquid sample and the vibration frequency of the sample dosing needle.

[0011] To achieve the above objective, this application adopts the following technical solution.

[0012] 1. A control device for producing microdroplets, wherein the control device includes a means for producing microdroplets. The microdroplet production means includes an asymmetric vibrating microdroplet generation mechanism and a first dynamic positioning member, the first dynamic positioning member being fixedly connected to the asymmetric vibrating microdroplet generation mechanism, The first dynamic positioning member is configured to position the asymmetric vibrating microdroplet generation mechanism, The aforementioned asymmetric vibrating microdroplet generation mechanism is configured to generate microdroplets using an asymmetric reciprocating motion method.

[0013] 2. The control device according to item 1, wherein the asymmetric vibrating microdroplet generation mechanism generates one microdroplet within one motion cycle.

[0014] 3. The asymmetric vibrating microdroplet generation mechanism includes a vibrating member, a vibrating mounting base, a sample dosing needle, and a drive controller. The control device according to claim 1, wherein the drive controller is electrically connected to the vibrating member and drives the vibrating mounting base to vibrate or swing asymmetrically in a reciprocating motion so that the sample dosing needle generates microdroplets.

[0015] 4. The control device according to item 3, wherein the sample-adding needle generates one microdroplet within one period of asymmetric reciprocating vibration or asymmetric reciprocating oscillation of the vibrating mounting base.

[0016] 5. The vibrating member includes a housing, a vibrator, and a vibrating output rod. The vibration output rod of the vibrating member provides power to the vibration mounting base. The vibration mounting base has a connection port, a pipe joint, and a sample dosing needle adapter. One end of the connection port is connected to the liquid supply conduit via a pipe fitting, and the other end is connected to the sample addition needle via a sample addition needle adapter. The control device according to item 3, wherein the central axis of the sample dosing needle is perpendicular to the axis of the vibration output rod.

[0017] 6. The control device according to item 5, wherein the asymmetric reciprocating motion is an asymmetric reciprocating vibration performed by the vibration mounting base in a direction along the central axis of the vibration output rod.

[0018] 7. The control device according to item 5, wherein the asymmetric reciprocating motion is an asymmetric oscillation of the vibration mounting base with the central axis of the vibration output rod as its axis.

[0019] 8. The vibration frequency of the vibrating member is 10 to 1000 Hz, preferably 50 to 200 Hz. Preferably, the vibration amplitude of the vibrating member is 0.1 to 5 mm, preferably 0.5 to 2 mm, the control device according to item 3 or 5.

[0020] 9. The oscillation frequency of the vibration mounting base is 10 to 1000 Hz, preferably 50 to 200 Hz. Preferably, the distance between the liquid discharge opening of the sample addition needle and the axis of the vibration output rod is 10 to 100 mm, preferably 30 to 80 mm, and the rocking angle width of the vibration mounting base is 0.05 to 10°, preferably 0.2 to 2°. The control device according to item 5.

[0021] 10. The vibration mounting base and the vibration output rod are connected by a coupling. The control device according to item 5.

[0022] 11. The vibration member 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. Preferably, the position sensor is one of a grating scale sensor, a capacitance type position sensor, a resistance type sensor, a current type sensor or a differential transformer type sensor. The control device according to item 3 or 5.

[0023] 12. The asymmetric vibration micro-droplet generation mechanism further includes a support fixing base for fixing the vibration member. . Preferably, the asymmetric vibration micro-droplet generation mechanism further includes a pump tube clamp base for clamping the liquid supply conduit. The control device according to item 5.

[0024] 13. There are a plurality of the connection ports, and the plurality of connection ports are provided at equal intervals inside the vibration mounting base. Preferably, the number of the connection ports is 1 to 96, preferably 2, 4, 8, or 12. The control device according to item 3 or 5.

[0025] 14. The asymmetric vibration micro-droplet generation mechanism further includes a connection guide structure. The vibration output rod of the vibration member is connected to the vibration mounting base through the connection guide structure to provide power to the vibration mounting base. The control device according to item 5.

[0026] 15. The control device according to item 14, wherein the connecting guide structure 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.

[0027] 16. The control device according to item 14, wherein the connecting guide structure includes a first bearing and a second bearing, one end of the vibration mounting base is connected to the vibration output rod through the first bearing, the other end of the vibration mounting base is connected to the second bearing, and the first bearing is a bearing having an axial locking edge.

[0028] 17. The control device according to claim 3 or 5, further comprising a sample dosing needle removal mechanism for automatically removing the sample dosing needle after microdroplets have been generated.

[0029] 18. The sample dosing needle has a conical tubular structure with open ends, one end being a liquid supply opening for tight insertion with the sample dosing needle adapter, and the other end being a liquid discharge opening for generating microdroplets, the inner diameter of the liquid discharge opening being 20-300 μm and the outer diameter being 150-600 μm. Preferably, the liquid storage volume of the sample-adding needle is in the range of 5 to 500 μL, preferably 20 to 60 μL, the control device according to item 5.

[0030] 19. When the vibrating mounting base performs the asymmetric reciprocating motion, the motion of the liquid dispensing portion of the sample dosing needle has one equilibrium point and two reflection points at both ends of the equilibrium point, and the position-time curve of the motion is configured to be asymmetric on both sides of either reflection point. Preferably, the control device according to claim 3 or 5, wherein the asymmetric waveform of the periodic motion of the liquid dispensing portion of the sample dosing 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.

[0031] 20. The control device according to item 3 or 5, wherein the vibrating member is configured as a mechanism for generating continuous or intermittent motion, and the vibrating member is one selected from an electromagnetic vibrating device, a piezoelectric ceramic vibrating device, an eccentric wheel vibrating device, a servo motor, a voice coil motor, and a galvanometer motor.

[0032] 21. The first dynamic positioning member includes a positioning member lifting displacement mechanism for controlling the lifting and lowering of the asymmetric vibrating microdroplet generation mechanism. Preferably, the control device according to item 1, wherein the first dynamic positioning member further includes a liquid level detection mechanism.

[0033] 22. The control device further includes fluid control means, The fluid control means includes a fluid drive device and a conduit, one end of the conduit is connected to the fluid drive device, and the other end of the conduit is connected to the asymmetric vibrating microdroplet generation mechanism. Preferably, the control device according to item 1, wherein the fluid drive device is used to set the flow rate and cause the sample addition needle to absorb and drain liquid.

[0034] 23. The control device further includes a second dynamic positioning member, the second dynamic positioning member used to fix and move the first opening container and the second opening container. Preferably, the control device according to item 22, wherein the drive controller provides power to the fluid drive device, the first dynamic positioning member and the second dynamic positioning member.

[0035] 24. 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 to 1000 μL, preferably 20 to 200 μL. Preferably, the first open container contains the first liquid. Preferably, the second open container is a two-dimensional flat-bottom sample cell array for stacking the generated microdroplets, and preferably, the second open container contains 24, 32, 96, or 384 flat-bottom sample cells of equal volume. Preferably, the control device according to item 23, wherein the second open container contains a second liquid.

[0036] 25. The control device according to item 22, wherein the fluid drive device is a pulsation-free drive pump, preferably a syringe pump, and more preferably one or more fluid drive devices.

[0037] 26. The control device according to item 22, wherein the conduit includes a liquid supply conduit and a liquid suction conduit, one end of the liquid suction conduit is connected to the fluid drive device via one valve port of an electric two-position three-way control valve, the other end of the liquid suction conduit is inserted into an oil reservoir, one end of the liquid supply conduit is connected to the fluid drive device via one valve port of an electric two-position three-way control valve, and the other end of the liquid supply conduit is connected to the asymmetric vibrating microdroplet generation mechanism.

[0038] 27. The control device according to item 1, further comprising a preparation means, the preparation means comprising a sample-adding needle rack, an oil removal mechanism, and a waste receiver, wherein the oil removal mechanism is located above the waste receiver, and the sample-adding needle is removed above the waste receiver.

[0039] 28. Using a fluid drive device, drive the conduit, the sample injection needle adapter, and the sample injection needle to fill the inside of the sample injection needle with the first liquid. A method for producing microdroplets using a control device according to any one of claims 1 to 27, comprising bringing a sample-dosing needle filled with a first liquid into contact with a second liquid, and generating microdroplets by driving a sample-dosing needle adapter to cause the sample-dosing needle to reciprocate asymmetrically below the liquid surface of the second liquid under the drive of a fluid drive device.

[0040] 29. Using a fluid drive device, the conduit, sample addition needle adapter, and sample addition needle are driven, and before filling the sample addition needle with the first liquid, the method further: a) The sample addition needle adapter is moved to the oil removal mechanism, the fluid drive device drives the liquid suction conduit to draw up the carrier liquid, and switches the electric two-position three-way switching valve, the fluid drive device discharges the carrier liquid from the sample addition needle adapter, and the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve and sample addition needle adapter are filled with carrier liquid and free of air bubbles, and the oil removal mechanism removes the excess carrier liquid discharged from the lower end opening of the sample addition needle adapter. b) Insert the sample addition needle into the sample addition needle adapter filled with carrier solution, and connect the sample addition needle to the sample addition needle adapter. c) The method according to claim 28, comprising moving the sample addition needle to the oil removal mechanism, switching the electric two-position three-way switching valve, and repeating step a) to ensure that the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve, sample addition needle adapter, and sample addition needle are filled with carrier liquid and free of air bubbles, and the oil removal mechanism removes excess carrier liquid discharged from the opening of the sample addition needle.

[0041] 30. The step of using a fluid drive device to drive the conduit, sample addition needle adapter, and sample addition needle, and filling the inside of the sample addition needle with the first liquid, The method according to claim 29, comprising moving the sample-adding needle of step c) above the liquid level of the first open container containing the first liquid, moving it downward, bringing the liquid outlet of the sample-adding needle into contact with and immersing it in the first liquid, switching the electric two-position three-way switching valve to draw the first liquid into the sample-adding needle and fill the inside of the sample-adding needle with the first liquid.

[0042] 31. The method according to item 30, wherein the volume of the first liquid drawn in from the liquid outlet of the sample-adding needle is less than the volume of the connection cavity of the sample-adding needle.

[0043] 32. The method according to claim 28, further comprising removing the sample addition needle after the preparation of a first liquid microdroplet is complete, and then reinserting the sample addition needle to prepare another first liquid microdroplet.

[0044] 33. The method according to item 28, wherein the sample-adding needle performs asymmetric reciprocating vibration or asymmetric oscillation below the liquid surface of the second liquid, and a fluid drive device is used to set the flow rate and discharge the first liquid from the sample-adding needle to generate microdroplets.

[0045] 34. The method according to item 28, wherein the sample-adding needle generates only one microdroplet within one period of asymmetric reciprocating motion below the liquid surface of the second liquid.

[0046] 35. The method according to item 28, characterized in that the first dynamic positioning member drives an asymmetric vibrating microdroplet generation mechanism to precisely position the sample dosing needle below the liquid surface of the second liquid, and during the asymmetric vibrating process, the average depth of the sample dosing needle inserted into the liquid surface is dynamically maintained in the range of 0 to 2.0 mm below the liquid surface, preferably in the range of 0 to 1.5 mm below the liquid surface.

[0047] As can be seen from the above technical proposal, the advantages and positive effects of the microdroplet horizontal vibration generation mechanism and microdroplet generation method provided by this application are as follows.

[0048] 1) Compared to micropipettes based on the principle of air pistons, the control device provided in this application has a liquid aspiration conduit, a liquid supply conduit, an electric two-position three-way switching valve, a sample docking needle adapter, and a sample docking needle, all of which are filled with carrier liquid. It can accurately aspirate and discharge nanoliter volumes of liquid samples using the principle of positive displacement. At the same time, because the volume of the liquid sample aspirated by the control device is smaller than the volume of the connection cavity of the sample docking needle, when creating microdroplets of different samples, the sample docking needle only needs to be changed according to the microdroplet preparation method, thereby reducing the complexity of experimental operations.

[0049] 2) The control device provided herein includes a control device for a second open container containing a second liquid, the resulting microdroplets settle and are stacked flat at the bottom of a flat-bottomed sample cell in the second open container, and the nanoliter volume of microdroplets is protected by the oily second liquid, thereby preventing evaporation of the microdroplets and simplifying the microdroplet transfer and storage steps, as well as the subsequent microdroplet imaging detection and analysis steps.

[0050] 3) In the control device provided by the present invention, the vibrating member of the microdroplet generation mechanism drives the liquid discharge opening of the sample-adding needle on the vibrating mounting stand to perform a periodic reciprocating motion with varying acceleration between two points below the oil phase surface or between two points straddling the oil phase surface. By adjusting only the flow velocity of the aqueous phase liquid in the sample-adding needle and the vibration frequency of the sample-adding needle, it is possible to produce microdroplets of uniform size and controllable volume.

[0051] 4) In the first dynamic positioning member provided by this application, the asymmetric vibrating microdroplet generation mechanism is fixedly connected, and under precise dynamic positioning conditions of the first dynamic positioning member, the asymmetric vibrating microdroplet generation mechanism drives a sample dosing needle to generate microdroplets in an asymmetric reciprocating motion manner below the liquid surface of the second liquid. This avoids secondary shear fracture of droplets caused by severe disturbances of the oil phase due to the inability of the micropipe to contact the liquid surface of the second liquid or the insertion depth being too deep, due to dynamic changes in the liquid surface, liquid dosing errors, sample dosing needle length errors, system assembly errors, etc., thereby effectively ensuring the stability, continuity, and consistency of the droplet generation system.

[0052] 5) In the control device provided by this application, the microdroplet generation mechanism is provided with a connecting guide structure that resists torque and radial forces, thereby avoiding the torque and radial forces that the inertial forces and gravity of the multiple sample dosing needles and the vibration mounting base apply to the vibration output rod during the array generation of microdroplets and the automatic mounting process of the sample dosing needles, and ensuring the precision of asymmetric reciprocating motion and the stability and uniformity of microdroplet generation.

[0053] 6) In the control device provided by this application, the microdroplet generation mechanism employs an asymmetric vibration method, ensuring that there is only one maximum velocity position or maximum cutting force position within one cycle, and that asymmetric processing defects of the sample docking needle do not affect droplet generation, thus providing high tolerance for processing defects in consumables and ensuring the reliability and uniformity of droplet production.

[0054] 7) In the control device provided by the present invention, the drive controller is a preset asymmetric motion The control program enables closed-loop feedback control using real-time position feedback signals from the position sensor, achieving precise asymmetrical reciprocating motion of the vibration mounting base and enabling high-precision, uniform generation of nanoliter droplets.

[0055] 8) By utilizing the microdroplet production method provided in this application, stable and continuous production of microdroplets with uniform size and controllable volume can be achieved simply by adjusting the flow rate of the liquid sample and the vibration frequency of the sample dosing needle using the control device described above. Various objects, features, and advantages of the present invention will become more apparent by describing in detail the following preferred embodiments of the invention with reference to the accompanying drawings. The drawings are illustrative of the present application and are not necessarily drawn to scale. In the drawings, the same reference numerals indicate the same or similar parts. [Brief explanation of the drawing]

[0056] [Figure 1] This is a schematic diagram of the structure of a control device for producing microdroplets provided in this application. [Figure 2] This is a schematic diagram of the three-dimensional structure of a microdroplet generation mechanism that vibrates asymmetrically along the central axis direction of the vibration output rod provided in this application, and is an enlarged view of reference numeral 1 in Figure 1. [Figure 3]This is a schematic cross-sectional view of a microdroplet generation mechanism that vibrates asymmetrically along the central axis direction of the vibration output rod provided in this application, and is an enlarged view of reference numeral 1 in Figure 1. [Figure 4] This is a schematic diagram of the three-dimensional structure of the asymmetrically oscillating microdroplet generation mechanism provided by this application, and is an enlarged view of reference numeral 1 in Figure 1. [Figure 5] This is a front view of the asymmetrically oscillating microdroplet generation mechanism provided by the present invention, and is an enlarged view of reference numeral 1 in Figure 1. [Figure 6] This is a schematic cross-sectional view of the asymmetrically oscillating microdroplet generation mechanism provided by the present invention, and is an enlarged view of reference numeral 1 in Figure 1. [Figure 7] This is a diagram showing the structure and control logic of the asymmetrically oscillating microdroplet generation mechanism provided by this application. [Figure 8] The following are the configurations of the liquid discharge portion of a sample dosing needle applied to the microdroplet generation mechanism provided by this application: (a) a 3D stereoscopic view of the sample dosing needle; (b) a cross-sectional view of the sample dosing needle; (c) an ideal structure in which the liquid discharge portion of the sample dosing needle is flat and has a uniform wall thickness; (d) a structure in which the liquid discharge portion of the sample dosing needle is flat, does not have a uniform wall thickness, and the sample dosing needle exhibits an asymmetrical structure; (e) a burr defect exists on the outside of the liquid discharge opening of the liquid discharge portion of the sample dosing needle; and (f) a burr defect exists on the inside of the liquid discharge opening of the liquid discharge portion of the sample dosing needle. [Figure 9] The curves of the asymmetric vibration of a combination of sinusoidal and sawtooth waves employed by a microdroplet generation mechanism in one specific embodiment provided by this application are (a) a vibration position-time curve and (b) a vibration velocity-time curve. [Figure 10] The curves of the asymmetric vibration of the combination of asymmetric sawtooth waves employed by a microdroplet generation mechanism in one specific embodiment provided herein are (a) a position-time vibration curve and (b) a velocity-time vibration curve. [Figure 11]The curves of the asymmetric vibration of a combination of sawtooth waves and square waves employed by a microdroplet generation mechanism in one specific embodiment provided herein are (a) a vibration position-time curve and (b) a vibration velocity-time curve. [Figure 12] The comparative embodiment of the present invention employs a symmetrical vibration position-time curve, which is (a) a sine wave and (b) a square wave. [Figure 13] This figure shows the result of the microdroplet generation mechanism generating microdroplets in one embodiment 1 of the present invention. [Figure 14] This figure shows the result of the microdroplet generation mechanism generating microdroplets in one embodiment 2 of the present invention. [Figure 15] This figure shows the results of generating microdroplets in Comparative Example 1 of the present invention, where the microdroplet generation mechanism employs a symmetrical vibration waveform. [Figure 16] This figure shows the results of generating microdroplets in Comparative Example 2 of the present invention, where the microdroplet generation mechanism employs a symmetrical vibration waveform. [Figure 17] This is a schematic diagram of the vibration position of the asymmetrically reciprocating microdroplet generation mechanism provided by this application. [Figure 18] This is a schematic diagram of the oscillation position of the asymmetrically oscillating microdroplet generation mechanism provided by this invention. [Figure 19] This is a schematic diagram of the vibration position of the asymmetrically reciprocating microdroplet generation mechanism provided by this application. [Figure 20] This is a schematic diagram of the vibration position of the asymmetrically oscillating microdroplet generation mechanism provided by this invention. [Figure 21] This is a schematic diagram of the structure of a control device for producing microdroplets provided in this application. [Figure 22] This is a schematic diagram of the droplets produced in Example 3. [Figure 23] This is a schematic diagram of the droplets produced in Example 4. [Figure 24] This is a schematic diagram of the droplets produced in Example 5. [Figure 25] This is a schematic diagram of the droplets produced in Example 6. [Figure 26]This is a schematic diagram showing how droplets generated by different flow rates of the first liquid sample in Examples 7-18 are stacked flat at the bottom of the container. [Figure 27] This is a schematic diagram showing the relationship between the volume of droplets generated in Examples 19-28 and the theoretical microdroplet volume. [Figure 28] This is a schematic diagram of droplets of different volumes generated from different concentrations of human genome samples in Example 29. [Figure 29] This is a schematic diagram showing the relationship between the digital PCR quantitative value after amplification using PCR in Example 29 and the nucleic acid sample concentration. [Modes for carrying out the invention]

[0057] Typical embodiments that embody the features and advantages of the present invention are detailed in the following description. The present invention may have various modifications in different embodiments, and it should be understood that the description and drawings are for illustrative purposes only and not to limit the scope of the present invention.

[0058] In the following description of different exemplary embodiments of the Application, the descriptions are made with reference to the drawings, which form part of the Application and illustrate different exemplary structures, systems, and steps in a plurality of exemplary embodiments of the Application. It should be understood that other specific solutions may be used for parts, structures, exemplary devices, systems, and steps, and that the structure and function may be modified without departing from the scope of the Application. Also, in this Specification, terms such as “upper end,” “lower end,” “between,” “side,” etc., may be used to describe different exemplary features and elements of the Application, but these terms are for convenience in this Specification and follow, for example, the exemplary orientation shown in the drawings. Nothing in this Specification should be understood as requiring a specific three-dimensional orientation of a structure to fall within the scope of the Application.

[0059] Figure 1 is a schematic diagram of the structure of a control device for producing microdroplets disclosed in this application, and Figures 2 to 7 are enlarged views of the asymmetric vibrating microdroplet generation mechanism 1 in Figure 1. In Figure 1, the control device includes a microdroplet production means, the microdroplet production means includes the asymmetric vibrating microdroplet generation mechanism 1 and a first dynamic positioning member 200, the first dynamic positioning member 200 is fixedly connected to the microdroplet generation mechanism 1.

[0060] The first dynamic positioning member 200 is configured to position the asymmetric vibrating microdroplet generation mechanism 1. The asymmetric vibrating microdroplet generation mechanism 1 is configured to generate microdroplets using an asymmetric reciprocating motion method. The control device further includes fluid control means, which includes a fluid drive device 500 and a conduit (not shown), one end of which is connected to the fluid drive device 500 and the other end of which is connected to the microdroplet generation mechanism 1. The fluid drive device 500 is used to set the flow rate and cause the sample addition needle 400 to absorb and drain liquid.

[0061] 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 drive device 500 via one valve port of an electric two-position three-way switching valve (not shown), and the other end of the liquid suction conduit 502 is inserted into an oil storage device (e.g., an oil storage bottle 504). One end of the liquid supply conduit 501 is connected to the fluid drive device 500 via one valve port of an electric two-position three-way switching valve, and the other end of the liquid supply conduit 501 is connected to the microdroplet generation mechanism 1.

[0062] The oil storage bottle 504 contains a carrier liquid 505, and the fluid drive device 500 fills the liquid suction conduit 502, liquid supply conduit 501, electric two-position three-way switching valve, sample addition needle adapter, and sample addition needle 400 with the carrier liquid 505.

[0063] The carrier liquid may be, for example, one or more of liquid alkanes, liquid esters, and fluorinated alkanes.

[0064] In one embodiment, only one microdroplet is generated within one oscillation cycle of the asymmetric vibrating microdroplet generation mechanism.

[0065] In one embodiment, the asymmetric reciprocating motion method of the asymmetric vibrating microdroplet generation mechanism may include asymmetric reciprocating vibration and asymmetric reciprocating oscillation. Within one period in which the vibrating mounting base vibrates or oscillates asymmetrically, the sample dosing needle generates only one microdroplet.

[0066] As shown in Figures 2 to 7, the microdroplet generation mechanism 1 includes a vibrating member 100, a connecting guide structure 105, a vibrating mounting base 300, a sample dosing needle 400, and a drive controller 600. The vibrating member 100 includes a housing 101, a vibrator 102, and a vibrating output rod 104. The vibration output rod 104 of the vibrating member 100 is connected to the vibration mounting base 300 via a connecting guide structure 105, and provides power to the vibration mounting base 300. The vibration mounting base 300 has a connection port 304, a pipe joint 302, and a sample dosing needle adapter 301. One end of the connection port 304 is connected to the liquid supply conduit 501 via a pipe fitting 302, and the other end is connected to the sample addition needle 400 via a sample addition needle adapter 301. The central axis of the sample-adding needle 400 is perpendicular to the axis of the vibration output rod 104. The drive controller 600 is electrically connected to the vibrating member 100 and drives the vibrating mounting base 300 to perform asymmetric reciprocating motion according to the asymmetric motion control program 602 so that the sample dosing needle generates microdroplets.

[0067] In one embodiment, the drive controller 600 is asymmetrical with respect to the vibration drive circuit 601. motionThe system includes a 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 vibrating member 100 and drives the vibrating mounting base 300 to perform asymmetric reciprocating motion according to the asymmetric reciprocating motion control program 602.

[0068] In one embodiment, the asymmetric reciprocating motion is an asymmetric reciprocating vibration performed by the vibration mounting base 300 in a direction along the central axis of the vibration output rod 104.

[0069] In one embodiment, the connecting guide structure 105 is a ball spline 105 including a spline shaft 1052 and a spline sleeve 1051, and both ends of the spline shaft 1052 are fixedly connected to the vibration output rod 104 and the vibration mounting base 300, respectively, and the connection between the spline shaft 1052 and the vibration output rod 104 is made via a connecting member 106. In one embodiment, the connecting member 106 is a screw.

[0070] In one embodiment, the vibrating member 100 is a vibrating member that provides power for asymmetric reciprocating motion to the vibrating mounting base 300 and can generate continuous or intermittent vibrations, and is selected from an electromagnetic vibrating device, a piezoelectric ceramic vibrating device, an eccentric wheel vibrating device, a servo motor, a voice coil motor, and a galvanometer motor. In the production of microdroplets, the vibration frequency and amplitude of the vibrating device used can be selected according to actual requirements. For example, the vibration frequency of the vibrating member 100 is 10 to 1000 Hz, preferably 50 to 200 Hz, and the vibration amplitude is 0.1 to 5 mm, preferably 0.5 to 2 mm.

[0071] The connecting guide structure 105 (ball spline) can restrain the movement of the vibration mounting base 300 so that it can only reciprocate in the direction of the central axis of the ball spline 105. Specifically, when the microdroplet generation mechanism of the present invention is actually applied, the vibration output rod 104 receives a radial force perpendicular to its axial direction generated by the weight of the vibration mounting base 300 and the sample dosing needle 400 it bears, and a torque force generated by the unbalanced weight at both ends of the vibration mounting base 300. This prevents the vibration output rod 104 or the ball spline 105 from vibrating in any direction other than the central axis, allowing the vibration mounting base 300 to reciprocate only in the direction of the central axis of the ball spline 105, thereby ensuring the stability and uniformity of microdroplet generation.

[0072] The other end of the connecting guide structure 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.

[0073] In one embodiment, the vibrating member 100 further includes a position sensor 103, and the drive controller 600 collects real-time position feedback signals from the position sensor 103 of the vibrating member 100 using a position signal acquisition module 604. Real-time position feedback signal and Asymmetric reciprocating motion control program to Real Thai Mu ratio comparison to position By feeding back to the position correction module 603 and adjusting the control parameters of the vibration drive circuit 601, closed-loop control of asymmetric reciprocating motion is achieved.

[0074] The vibrating member 100 of the present invention periodically performs asymmetric reciprocating motion and has one equilibrium point EP and two reflecting points RP1 and RP2 at both ends of the equilibrium point. Asymmetric reciprocating motion is defined as one vibration period, which is the time it takes to go from the reflecting point RP1, through the equilibrium point EP to the reflecting point RP2, and then return from the reflecting point RP2, again through the equilibrium point EP to the reflecting point RP1. Figure 17 is a schematic diagram of the vibration position of the asymmetric reciprocating microdroplet generation mechanism provided by the present invention, where EP is located at the center of RP1 and RP2, that is, the distance between EP and RP1 is equal to the distance between EP and RP2. Figure 18 is a schematic diagram of the vibration position of the asymmetric oscillating microdroplet generation mechanism provided by the present invention, where EP is located at the center of RP1 and RP2, that is, the distance between EP and RP1 is equal to the distance between EP and RP2. Figure 19 is a schematic diagram of the vibration position of the asymmetrically reciprocating microdroplet generation mechanism provided by the present invention, where the distance between EP and RP1 is not equal to the distance between EP and RP2. Figure 20 is a schematic diagram of the vibration position of the asymmetrically oscillating microdroplet generation mechanism provided by the present invention, where the distance between EP and RP1 is not equal to the distance between EP and RP2.

[0075] In one embodiment, the asymmetrically reciprocating vibrating member described in this application has EP located at the center of RP1 and RP2, with the position at the equilibrium point EP as 0, time on the horizontal axis, and the vibration position on the vertical axis to form a curve, and the waveform curve of vibration position versus time is asymmetric on both sides of an arbitrary reflection point (RP1 or RP2) (Figures 9, 10, and 11). Due to this periodic asymmetric reciprocating motion, the liquid discharge opening of the sample docking needle reaches its maximum motion speed at only one time point or time period within two half-cycles of motion, and the shear force of the oil phase action acting on the liquid discharge opening is different, that is, in the process of the liquid discharge opening moving periodically, the liquid discharge opening has only one point of maximum shear force application within one cycle. In one embodiment, the asymmetrically reciprocating vibrating member described in this application has a distance between EP and RP1 that is not equal to the distance between EP and RP2, and due to the periodic asymmetric reciprocating motion, the liquid discharge opening of the sample-adding needle reaches its maximum motion speed (Figures 19 and 20) at only one point in time or one time period within two half-cycles of motion, that is, in the process of the liquid discharge opening moving periodically, the liquid discharge opening has only one maximum shear force acting section within one cycle. Compared to the asymmetric reciprocating motion of the present invention, droplets can also be generated by symmetric reciprocating vibration below the liquid surface. However, the vibrating member has two maximum points of action in one full cycle, meaning that the vibrating member has the potential to generate droplets in both half-cycles. In other words, it may generate one small droplet per half-cycle or one large droplet twice the volume in the entire cycle. Therefore, there is a great deal of uncertainty in the size of the droplets. Furthermore, the opening of a micropipe processed by injection molding or the like cannot be made perfectly symmetrical in practice (as shown in Figures 8d, 8e, and 8f), and there are differences in the size of the droplets generated in the two half-cycles. Accordingly, the present invention solves the problem of non-uniform droplet generation in the symmetric reciprocating vibration and the excessively high demands on processing symmetry, precision, and defects of the liquid discharge opening by using asymmetric reciprocating vibration, thereby achieving highly controllable and uniform droplet generation.

[0076] 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 vibrating member 100 can only reciprocate along the central axis direction of the vibration output rod 104. In one embodiment, the asymmetric reciprocating motion is an asymmetric oscillation with the central axis of the vibration output rod 104 as the axis. Figures 2 and 3 show schematic diagrams of a microdroplet generation mechanism that reciprocates asymmetrically along the central axis direction of the vibration output rod.

[0077] In one embodiment, the asymmetric reciprocating motion is an asymmetric oscillation of the vibration mounting base with the central axis of the vibration output rod as its axis, and as shown in Figures 4 to 7, the microdroplet generation mechanism is schematic in which the vibration mounting base oscillates asymmetrically with the central axis of the vibration output rod 104 as its axis. The vibrating member 100 includes a housing 101, a vibrator 102, a position sensor 103, and a vibration output rod 104. The vibration output rod 104 of the vibrating member 100 is connected to the vibration mounting base 300 via a connecting guide structure 105 and a connecting member 106, and provides the vibration mounting base 300 with power for the reciprocating motion. The central axis of the sample dosing needle 400 is perpendicular to the axis of the vibration output rod 104.

[0078] The connecting guide structure 105 can restrain the movement of the vibration mounting base 300, which is fixed to the vibration output rod 104, so that it can only swing asymmetrically around the central axis of the vibration output rod 104. The connecting guide structure 105 includes a first bearing 1053 and a second bearing 1054, and one end of the vibration mounting base 300 passes through the first bearing 1053. Vibration output rod 104The other end of the vibration mounting base 300 is connected to the second bearing 1054, and the first bearing 1053 is a bearing having an axial locking edge. In one embodiment, the connecting member 106 is a coupling. The first bearing 1053 having an axial locking edge prevents the vibration mounting base 300 from being displaced in the axial direction of the vibration output rod 104, and by using the first bearing 1053 and the second bearing 1054 in combination, the structural stability of the vibrating member and the stability and uniformity of microdroplet generation are guaranteed.

[0079] The vibrating member 100 is a mechanism that provides power for asymmetric reciprocating motion to the vibrating mounting base 300 and can generate continuous or intermittent motion, and is selected from an electromagnetic vibrating device, a piezoelectric ceramic vibrating device, an eccentric wheel vibrating device, a servo motor, a voice coil motor, and a galvanometer motor. In the production of microdroplets, the vibration frequency and amplitude of the vibrating device, or the oscillation amplitude and oscillation, can be selected according to actual requirements. In one embodiment, the vibration frequency is 10 to 1000 Hz, preferably 50 to 200 Hz. In one embodiment, the vibration amplitude of the vibrating device is 0.1 to 5 mm, preferably 0.5 to 2 mm. In one embodiment, the oscillation frequency is 10 to 1000 Hz, preferably 50 to 200 Hz. In one embodiment, the distance between the liquid discharge opening of the sample addition needle and the axis of the vibration output rod 104 is 10 to 100 mm, preferably 30 to 80 mm, and the swing angle range of the vibration mounting base is 0.05 to 10°, preferably 0.2 to 2°.

[0080] The vibration mounting base 300 of this application is a fixing device for the sample dosing needle 400, and generates microdroplets by transmitting the driving force of the vibrating member 100 to the liquid loaded in the sample dosing needle 400. The central axis of the sample dosing needle 400 is perpendicular to the axis of the vibration output rod 104.

[0081] Specifically, the vibration mounting base 300 has a connection port 304, one end of which is connected to the liquid supply conduit 501 via a pipe joint 302, and the other end of which is connected to the sample addition needle 400 via a sample addition needle adapter 301.

[0082] The connection port 304 is a through groove that serves to connect the liquid supply conduit 501 and the sample addition needle 400. Depending on the actual application needs, there may be one or more connection ports 304, for example, 1 to 96. Multiple connection ports 304 are provided at equal intervals inside the vibration mounting base 300. Preferably, there may be 2, 4, 8, or 12 connection ports 304.

[0083] As shown in Figures 8a and 8b, the sample addition needle 400 has a conical tubular structure with open ends. One end is a liquid supply opening 401 for tight insertion with the sample addition needle adapter 301, maintaining an airtight connection between the sample addition needle 400 and the sample addition needle adapter 301. The other end is a liquid discharge section 402 of the sample addition needle, which includes a liquid discharge opening 403 for generating microdroplets. The inner diameter of the liquid discharge opening 403 of the sample addition needle is 20 to 300 μm, and its outer diameter is 150 to 600 μm. The center of the sample addition needle is a liquid storage cavity 404 for storing the first liquid 800. The volume range of the liquid storage cavity 404 of the sample addition needle 400 is 5 to 500 μL, preferably 20 to 60 μL. Figures 8c-8f show the configuration of the liquid discharge section 402 of the sample dosing needle 400, which is adapted to the asymmetric reciprocating motion mechanism for microdroplet generation provided in this application. Here, Figure 8c shows the configuration of an ideally machined, burr-free, symmetrical liquid discharge section. By utilizing the asymmetric reciprocating vibration of the microdroplet generation mechanism of this application, defects in the configuration of the liquid discharge section 402 of the sample dosing needle can be tolerated, including asymmetry (Figure 8d), the presence of burrs on the outside of the liquid discharge opening (Figure 8e), and the presence of burrs on the inside of the liquid discharge opening (Figure 8f). The size of these machining defects is on the order of tens to hundreds of microns, which cannot be completely avoided under conventional machining process conditions and adversely affect the uniformity of droplet formation.

[0084] As shown in Figures 9 to 11, the position of the liquid discharge opening of the sample-adding needle 400 oscillates from side to side at the vibration center position, and the time-dependent change curve of the position of the liquid discharge opening 403 can exhibit one of several forms, such as a sine wave, sawtooth wave, trapezoidal wave, triangular wave, or square wave, or a superposition of multiple of the above waveforms. In one embodiment, the time-dependent change curve of the position of the liquid discharge opening of the sample-adding needle 400 is a combination of a sine wave and a sawtooth wave (shown in Figure 9a), and its periodic velocity-time curve is as shown in Figure 9b. In another embodiment, the time-dependent change curve of the position of the liquid discharge opening of the sample-adding needle 400 is a combination of a short-period sawtooth wave and a long-period sawtooth wave, as shown in Figure 10a, and its periodic velocity-time curve is as shown in Figure 10b. In one embodiment, the time-dependent change curve of the position of the liquid discharge opening of the sample-adding needle 400 is a combination of a rectangular wave and a sawtooth wave (shown in Figure 11a), and its periodic velocity-time curve is as shown in Figure 11b.

[0085] The characteristics of the waveform described above are that the velocity change curves of the liquid discharge opening of the sample-adding needle 400 are asymmetrical, with a maximum instantaneous velocity or maximum velocity interval greater than the average velocity within a single vibration cycle. The liquid discharge portion of the sample-adding needle 400 generates one droplet within one reciprocating vibration period due to the action of the asymmetrical reciprocating motion, and the generation of this droplet is controlled by the maximum instantaneous velocity or maximum velocity interval within the reciprocating vibration period, thus providing extremely high stability and reliability.

[0086] In one embodiment, the vibration generation mechanism of the present invention further includes a support and fixing base 120 for fixing the vibrating member 100 and for facilitating automated operations such as loading, moving, and droplet formation of a sample dosing needle.

[0087] In one embodiment, the vibration generation mechanism of the present application further includes a pump tube clamp base 303 (see Figures 3, 4, 5, 6, and 7) for clamping the liquid supply conduit 501. Specifically, the pump tube clamp base 303 can hold the liquid supply conduit 501 vertically during use, ensuring the stability of the liquid supply and ensuring that the generated microdroplets are stable and uniform.

[0088] In one embodiment, the first dynamic positioning member 200 includes a positioning member lifting and displacement mechanism 201, which is used to control the lifting and lowering and precise positioning of the microdroplet generation mechanism 1.

[0089] The first dynamic positioning member 200 further includes a liquid level detection mechanism 202 to assist the first dynamic positioning member 200 in accurately positioning the opening of the sample addition needle to a constant depth below the liquid level 702 of the second liquid located in the second opening container 701. When the liquid level changes due to the continuous generation of droplets or when there is a liquid level error due to a volume error in the addition liquid of the second liquid, the height position is dynamically adjusted under the control of the control device to ensure long-term stability and uniformity of droplet generation.

[0090] The liquid level detection mechanism 202 may be, for example, a high-resolution CCD camera, a reflective photoelectric sensor, or a capacitive distance sensor.

[0091] In one embodiment, the control device further includes a second dynamic positioning member 900, the second dynamic positioning member 900 being a precision two-axis horizontal translation stage for fixing and moving the first and second opening containers.

[0092] In one embodiment, the drive controller 600 not only drives and controls the asymmetric vibrating microdroplet generation mechanism 1, but also simultaneously drives and controls the fluid drive device 500, the first dynamic positioning member 200, and the second dynamic positioning member 900, and operates in cooperation with the asymmetric vibrating microdroplet generation mechanism 1.

[0093] In one embodiment, the first open container 801 (Figures 1 and 21) contains the first liquid 800.

[0094] The first liquid may be, for example, a PCR (Polymerase Chain Reaction) reagent, an RT-PCR (Reverse Transcription PCR) reagent, a microbial suspension, or a cell suspension.

[0095] The first open container is a single storage tank, a one-dimensional storage tank array, or a two-dimensional storage tank array, preferably an 8-tube PCR tube, more preferably a standard 96 or 384-well ELISA plate or PCR plate, and the bottom of the storage tank may be flat, round, or conical, with a volume of 10 to 1000 μL, preferably 20 to 200 μL.

[0096] For example, the volume of each first open container may 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.

[0097] As shown in Figures 1 and 21, the second open container 701 is pre-filled with a second liquid 700, the specific gravity of the second liquid being less than that of the first liquid, and the second open container is a two-dimensional flat-bottom sample cell array for stacking the generated microdroplets, preferably containing 24, 32, 96, or 384 flat-bottom sample cells of equal volume, the shape of which the flat-bottom sample cells are rectangular or circular.

[0098] The second liquid is one or more liquid alkanes or liquid esters containing a surfactant, and the surfactant is one or more of the following: Triton surfactants, Tween surfactants, Nonidet surfactants, Pluronic surfactants, Spun surfactants, Brij surfactants, IGEPAL surfactants, and ABIL EM / WE surfactants.

[0099] The Triton-based surfactant may be, for example, Triton X-100, Triton X-405, or Triton X-114.

[0100] The aforementioned twine-type surfactant may be, for example, twine 40, twine 60, twine 65, twine 80, twine 85, etc.

[0101] Nonidet surfactants may be, for example, P40.

[0102] The Pluronic surfactant may be, for example, Pluronic F-68.

[0103] The span-type surfactant may be, for example, span 20, span 40, span 60, span 80, span 85, etc.

[0104] Brij surfactants may include, for example, Brij30, Brij35, Brij58, Brij97, etc.

[0105] IGEPAL-based surfactants may include, for example, IGEPAL CO-630, IGEPAL CO-730, IGEPAL CO-520, IGEPAL CO-897, and the like.

[0106] ABIL EM / WE surfactants may include, for example, ABIL EM 97 and ABIL WE 09.

[0107] In one embodiment, the sample-adding needle adapter has a conical tubular structure with one end open, and the sample-adding needle adapter is used to insert the sample-adding needle. Preferably, the sample-adding needle adapter includes, from top to bottom, a liquid supply conduit connection, a connection cavity, and a liquid outlet. Preferably, the inner diameter of the connection cavity is 0.5 to 1.5 mm, preferably 1 mm. The outer diameter of the aforementioned connection cavity is 2 to 4 mm, preferably 2 to 3 mm. The length of the connection cavity is 10 to 50 mm, preferably 15 to 25 mm.

[0108] In one embodiment, the number of sample-adding needle adapters is 1 to 96, preferably 2, 4, 8, or 12.

[0109] In one embodiment, the fluid drive device is a pulsation-free drive pump, preferably a syringe pump.

[0110] The number of fluid drive devices is one or more.

[0111] In one embodiment, the liquid supply conduit 501, liquid suction conduit 502, electric two-position three-way switching valve, and sample addition needle adapter 301 are filled with carrier liquid 505 and free of air bubbles.

[0112] In one embodiment, the control device further includes preparation means (not shown), which include a sample-adding needle rack, an oil removal mechanism, a sample-adding needle removal mechanism, and a waste receiver, wherein the oil removal mechanism is located above the waste receiver, and the sample-adding needle is removed above the waste receiver.

[0113] The sample-adding needle rack, oil removal mechanism, sample-adding needle removal mechanism, and waste receiver are not limited in this application and can be selected as needed by those skilled in the art. For example, the oil removal mechanism may be made of sponge.

[0114] This application uses the fluid drive device to drive the conduit, the sample dosing needle adapter, and the sample dosing needle. Inside the sample insertion needle The first step is to fill with the liquid, The present invention provides a method for producing microdroplets using the control device described above, which includes the steps of: placing the opening of a sample-dosing needle filled with a first liquid below the liquid surface of a second liquid to maintain contact with the second liquid; and, under the drive of a fluid drive device, the microdroplet generation mechanism drives a sample-dosing needle adapter to cause the sample-dosing needle to reciprocate asymmetrically below the liquid surface of the second liquid, thereby generating microdroplets of the first liquid.

[0115] In one embodiment, the sample-adding needle vibrates or oscillates asymmetrically below the liquid surface of the second liquid, and the fluid drive device generates microdroplets by setting the flow rate and discharging the first liquid from the sample-adding needle.

[0116] In one embodiment, the sample-adding needle generates only one microdroplet within one cycle of asymmetric reciprocating motion below the liquid surface of the second liquid.

[0117] In one embodiment, the method may further include one or more of the following steps before using the fluid drive device to drive the conduit, the sample-adding needle adapter, and the sample-adding needle and filling the inside of the sample-adding needle with the first liquid. a) The sample addition needle adapter is moved to the oil removal mechanism, the fluid drive device drives the liquid suction conduit to draw up the carrier liquid, and switches the electric two-position three-way switching valve, the fluid drive device discharges the carrier liquid from the sample addition needle adapter, and the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve and sample addition needle adapter are filled with carrier liquid and free of air bubbles, and the oil removal mechanism removes the excess carrier liquid discharged from the lower end opening of the sample addition needle adapter. b) Insert the sample addition needle into the sample addition needle adapter filled with carrier solution, and connect the sample addition needle to the sample addition needle adapter. c) Move the sample addition needle to the oil removal mechanism, switch the electric two-position three-way switching valve, and repeat step a) to fill the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve, sample addition needle adapter, and sample addition needle with carrier liquid and free of air bubbles, and remove any excess carrier liquid discharged from the opening of the sample addition needle by the oil removal mechanism.

[0118] In one embodiment, the step of using a fluid drive device to drive the conduit, sample addition needle adapter, and sample addition needle, and filling the inside of the sample addition needle with a first liquid is: The procedure may also include moving the sample-adding needle from step c) above the liquid level of the first open container containing the first liquid, moving it downwards to bring the liquid outlet of the sample-adding needle into contact with and immerse it in the first liquid, switching the electric two-position three-way switching valve to draw the first liquid into the sample-adding needle and fill the inside of the sample-adding needle with the first liquid.

[0119] In one embodiment, the method is a) The sample addition needle adapter is moved to the oil removal mechanism, the fluid drive device drives the liquid suction conduit to draw up the carrier liquid, and switches the electric two-position three-way switching valve, the fluid drive device discharges the carrier liquid from the sample addition needle adapter, and the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve and sample addition needle adapter are filled with carrier liquid and free of air bubbles, and the oil removal mechanism removes the excess carrier liquid discharged from the lower end opening of the sample addition needle adapter. b) Insert the sample addition needle into the sample addition needle adapter filled with carrier solution, and connect the sample addition needle to the sample addition needle adapter. c) Move the sample addition needle to the oil removal mechanism, switch the electric two-position three-way switching valve, and repeat step a) to ensure that the carrier liquid is filled into the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve, sample addition needle adapter, and sample addition needle, and that there are no air bubbles, and that the oil removal mechanism removes the excess carrier liquid discharged from the opening of the sample addition needle. d) Move the sample-adding needle from step c) above the liquid level of the first open container containing the sample, and move it downward so that the liquid outlet of the sample-adding needle comes into contact with and is immersed in the first liquid, switch the electric two-position three-way switching valve, and repeat step a) to draw the first liquid into the sample-adding needle. e) Move the sample-adding needle from step d) out of the first open container, move it above the second open container containing the second liquid, and move it downward so that the liquid outlet of the sample-adding needle comes into contact with and is immersed in the second liquid. f) The first liquid inside the sample-doping needle is discharged from the liquid outlet of the sample-doping needle by the drive of a fluid drive device, and the microdroplet generation mechanism drives the sample-doping needle adapter to cause the sample-doping needle to vibrate or oscillate asymmetrically below the liquid surface of the second liquid, thereby detaching the discharged first liquid from the liquid outlet of the sample-doping needle and generating microdroplets.

[0120] In one embodiment, the volume of the sample drawn in from the liquid outlet of the sample-adding needle is the volume of the first liquid drawn in and supplied by the liquid supply. conduit To prevent contamination, the volume is smaller than the connection cavity of the sample dosing needle.

[0121] In one embodiment, the method further includes removing the sample-doping needle after the preparation of the first liquid microdroplets is complete, and then inserting another sample-doping needle according to steps b) to f) to prepare another first liquid microdroplet.

[0122] This invention provides a method for producing microdroplets using the above-described apparatus. The vibrating member of the microdroplet generation mechanism drives the liquid discharge opening of the sample-adding needle on the vibrating mounting stand to perform a periodic reciprocating motion with varying acceleration between two points below the oil phase surface or between two points straddling the oil phase surface. Therefore, by simply adjusting the flow velocity of the aqueous phase liquid in the sample-adding needle and the vibration frequency of the sample-adding needle, it is possible to produce microdroplets of uniform size and controllable volume.

[0123] Example 1 As shown in Figure 1, the control device includes a microdroplet production means, which includes an asymmetric vibrating microdroplet generation mechanism 1 and a first dynamic positioning member 200, the first dynamic positioning member 200 being fixedly connected to the microdroplet generation mechanism 1, and the first dynamic positioning member 200 being configured to position the asymmetric vibrating microdroplet generation mechanism 1. The asymmetric vibrating microdroplet generation mechanism 1 is configured to generate microdroplets using an asymmetric reciprocating motion method. The control device may further include fluid control means (not shown), which includes a fluid drive device 500, a liquid supply conduit 501, a liquid suction conduit 502, and an electric two-position three-way switching valve, wherein one valve port of the electric two-position three-way switching valve is connected to the fluid drive device 500, a second valve port is connected to one end of the liquid supply conduit 501, a 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 microdroplet generation mechanism 1.

[0124] The first dynamic positioning member 200 includes a positioning member lifting and displacement mechanism 201, and the microdroplet generation mechanism 1 is fixedly connected to the positioning member lifting and displacement mechanism 201 of the first dynamic positioning member 200.

[0125] The first dynamic positioning member 200 may further include a liquid level detection mechanism 202 to assist the first dynamic positioning member in accurately positioning the opening of the sample dosing needle to a depth of 0.3 mm below the liquid level 702 (Figure 21) of the second liquid located in the second opening container 701.

[0126] The control device further includes a second dynamic positioning member 900 for fixing and moving the first opening container 801 and the second opening container 701.

[0127] The first opening container 801 is a single liquid storage tank, and the first open container 801 contains the first liquid. The second open container 701 is a two-dimensional flat-bottom sample cell array for stacking the generated microdroplets. The control device may further include preparation means (not shown), which include a sample-adding needle rack, an oil removal mechanism (sponge), a sample-adding needle removal mechanism, and a waste receiver, wherein the oil removal mechanism (sponge) is located above the waste receiver, and the sample-adding needle is removed above the waste receiver.

[0128] Here, the microdroplet generation mechanism includes a vibrating member 100, a connecting guide structure 105, a vibrating mounting base 300, a sample dosing needle 400, and a drive controller 600, as shown in Figure 7.

[0129] As shown in Figures 2 and 3, the microdroplet generation mechanism in this embodiment vibrates asymmetrically along the central axis direction of the vibration output rod. The microdroplet generation mechanism includes a vibrating member, a connecting guide structure 105, a support fixing base 120, a vibration mounting base 300, a sample dosing needle 400, and a pump tube clamp base 303. The vibrating member 100 includes a housing 101, a vibrator 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 connecting guide structure 105 via a connecting member 106, and the other end of the connecting guide structure 105 is connected to the vibration mounting base 300. The central axis of the sample dosing needle 400 is perpendicular to the axis of the vibration output rod 104. The support fixing base 120 is used to fix the vibrating member 100.

[0130] The connecting guide structure 105 is a ball spline including a spline shaft 1052 and a spline sleeve 1051, with both ends of the spline shaft 1052 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 made by a connecting member 106, which is a screw. The position sensor 103 is partially connected to the vibrator and moves with the vibrator or vibration output rod, and the other part is fixed to the spline sleeve 1051, allowing for precise detection of displacement during vibration.

[0131] The vibrating member 100 is a vibration motor, the vibration frequency of the vibrator 102 is 100 to 200 Hz, the vibration amplitude range is 0.1 to 5 mm, and in actual application, the amplitude range is 0.5 to 1.5 mm.

[0132] The vibration mounting base 300 has connection ports 304, one end of which is connected to the liquid supply conduit 501 via a pipe fitting 302, and the other end of which is connected to the sample addition needle 400 via a sample addition needle adapter 301. There are eight connection ports 304, which are arranged at equal intervals inside the vibration mounting base 300. The pump tube clamp base 303 is configured to clamp the liquid supply conduit 501 in order to hold the liquid supply conduit 501 vertically.

[0133] The drive controller 600 is asymmetrical with respect to the vibration drive circuit 601. motionThe system includes a 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 vibrating member 100 and drives the vibrating mounting base 300 to perform asymmetric reciprocating motion according to the asymmetric reciprocating motion control program 602. The drive controller 600, using the position signal acquisition module 604, collects real-time position feedback signals from the position sensor 103 of the vibrating member 100, compares them in real time with the asymmetric reciprocating motion control program, and feeds them back to the position correction module 603, thereby adjusting the control parameters of the vibration drive circuit 601 and achieving closed-loop control of asymmetric reciprocating motion.

[0134] The drive controller 600 is further electrically connected to the first dynamic positioning member 200, the second dynamic positioning member 900, and the fluid drive device 500, providing power to each. If the liquid level changes due to the continuous generation of droplets or if there is a liquid level error due to a volume error in the added liquid of the second liquid, the drive controller 600 controls the first dynamic positioning member 200 to dynamically adjust its height, ensuring long-term stability and uniformity of droplet generation.

[0135] A method for producing microdroplets using the above-described control device may include the following steps. a) The sample addition needle adapter is moved to the sponge, the syringe pump drives the liquid suction conduit to draw up the carrier liquid, the electric two-position three-way switching valve is switched, the syringe pump discharges the carrier liquid from the sample addition needle adapter, the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve and sample addition needle adapter are filled with carrier liquid and free of air bubbles, and the sponge removes the excess carrier liquid discharged from the lower end opening of the sample addition needle adapter. The carrier liquid is a second liquid that is incompatible with the sample liquid, and the carrier liquid is an oil mainly composed of hydrocarbons, silicon or fluorocarbons, such as mineral oil, tetradecane, or FC40. b) Insert the sample addition needle into the sample addition needle adapter filled with carrier liquid, and connect the sample addition needle to the sample addition needle adapter. Here, the liquid storage volume of the sample addition needle is 60 μL, the material used to make the sample addition needle 400 is polypropylene (PP, with a contact angle of 88° in pure aqueous solution), the inner diameter of the liquid discharge opening of the sample addition needle 400 is 120 μm, and the outer diameter is 400 μm. c) Move the sample dosing needle to the sponge, switch the electric two-position three-way switching valve, and repeat step a) to fill the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve, sample dosing needle adapter, and sample dosing needle with carrier liquid and ensure there are no air bubbles, and remove any excess carrier liquid discharged from the opening of the sample dosing needle with the sponge. d) Move the sample-adding needle from step c) above the liquid level in the first open container containing the first liquid, and move it downward so that the liquid outlet of the sample-adding needle comes into contact with and is immersed in the aqueous solution (first liquid). Switch the electric two-position three-way switching valve and repeat step a) to draw 20 μL of the first liquid into the sample-adding needle. e) Move the sample-adding needle from step d) out of the first opening container and move it above the second opening container containing the mineral oil (second liquid), and the first dynamic Positioning The component is driven downwards by an asymmetric vibrating microdroplet generation mechanism, causing the liquid outlet of the sample-adding needle to come into contact with and immerse itself in the mineral oil at a depth of 0.3 mm below the liquid surface. f) The first liquid in the sample injection needle is discharged from the liquid outlet of the sample injection needle under the drive of the syringe pump, and the drive controller 600 enables drive control in which the sample injection needle reciprocates asymmetrically and periodically below or across the liquid surface of the second liquid, and the vibration motor in the microdroplet generation mechanism drives the sample injection needle adapter to cause the liquid discharge opening of the sample injection needle to reciprocate asymmetrically, and the position-time waveform of the motion is a combination of sine wave and sawtooth wave as shown in Figure 9, the vibration width is 1.2 mm, the vibration frequency is 100 Hz, the flow rate of the microsyringe is 100 nL / s, and the injection volume is 20 μL. Using the above parameter conditions, a 1 nL microdroplet is produced, and the microimaging of the microdroplet is as shown in Figure 13, and the CV of the volume of the droplet is 1.8%.

[0136] Example 2 As shown in Figure 1, the control device includes a microdroplet production means, which includes an asymmetric vibrating microdroplet generation mechanism 1 and a first dynamic positioning member 200, the first dynamic positioning member 200 being fixedly connected to the microdroplet generation mechanism 1, and the first dynamic positioning member 200 being configured to position the asymmetric vibrating microdroplet generation mechanism 1.

[0137] The asymmetric vibrating microdroplet generation mechanism 1 is configured to generate microdroplets using an asymmetric reciprocating motion method.

[0138] The control device further includes a fluid control means (not shown), the fluid control means including a fluid drive device 500, a liquid supply conduit 501, a liquid suction conduit 502, and an electric two-position three-way switching valve, wherein one valve port of the electric two-position three-way switching valve is connected to the fluid drive device 500, a second valve port is connected to one end of the liquid supply conduit 501, a 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 microdroplet generation mechanism 1.

[0139] The first dynamic positioning member 200 includes a positioning member lifting and displacement mechanism 201, and the microdroplet generation mechanism 1 is fixedly connected to the positioning member lifting and displacement mechanism 201 of the first dynamic positioning member 200.

[0140] As shown in Figure 21, the first dynamic positioning member 200 further includes a liquid level detection mechanism 202 to assist the first dynamic positioning member in accurately positioning the opening of the sample dosing needle to 0.3 mm below the liquid level 702 of the second liquid located in the second opening container 701. The liquid level detection mechanism is a high-resolution CCD camera that images the insertion process of the sample dosing needle into the liquid surface in real time and determines the distance between the sample dosing needle and the liquid surface and the insertion state by image analysis. The high-resolution CCD camera assists the first dynamic positioning member in accurately positioning the opening of the sample dosing needle to a constant depth below the liquid level of the second liquid located in the second opening container. If the liquid level height changes due to the continuous generation of droplets or if there is a liquid level height error due to a volume error in the second liquid dosing solution, the high-resolution CCD camera assists the first dynamic positioning member in dynamically adjusting the height position under the control of the control device, thereby ensuring long-term stability and uniformity of droplet generation.

[0141] The control device further includes a second dynamic positioning member 900 for fixing and moving the first opening container 801 and the second opening container 701.

[0142] The first open container 801 is a single liquid storage tank, and the first open container 801 contains the first liquid 800. The second open container 701 is a two-dimensional flat-bottom sample cell array for stacking the generated microdroplets. The control device further includes preparation means (not shown), which include a sample-adding needle rack, an oil removal mechanism (sponge), a sample-adding needle removal mechanism, and a waste receiver, wherein the oil removal mechanism (sponge) is located above the waste receiver, and the sample-adding needle is removed above the waste receiver.

[0143] Here, the microdroplet generation mechanism includes a vibrating member 100, a connecting guide structure 105, a vibrating mounting base 300, a sample dosing needle 400, and a drive controller 600, as shown in Figure 7.

[0144] As shown in Figures 4 to 7, the microdroplet generation mechanism in this embodiment drives the vibration mounting base 300 to oscillate asymmetrically around the central axis of the vibration output rod 104. The microdroplet generation mechanism includes a vibrating member 100 and a connecting guide structure 105. , shake The system includes a movable mounting base 300, a sample dosing needle 400, a support fixing base 120, and a pump tube clamp base 303. The vibrating member 100 includes a housing 101, a vibrator 102, a position sensor 103, and a vibrating output rod 104. One end of the vibrating output rod 104 is connected to the housing 101, and the other end is connected to the vibrating mounting base 300 via a connecting guide structure 105 and a connecting member 106. The central axis of the sample dosing needle 400 is perpendicular to the axis of the vibrating output rod 104.

[0145] The connecting guide structure 105 includes a first bearing 1053 and a second bearing 1054, and one end of the vibration mounting base 300 passes through the first bearing 1053. Connecting material 106 The other end of the vibration mounting base 300 is connected to the second bearing 1054, where the first bearing 1053 is a bearing having an axial locking edge. The connecting member 106 is a coupling.

[0146] The vibrating member 1 00 This is a galvanometer motor, and the vibration frequency of the vibrator 102 is 100-500 Hz, with a vibration amplitude range of 0.1-5 mm. In actual applications, the vibration frequency is 100-200 Hz, with a amplitude range of 0.5-1.5 mm.

[0147] The vibration mounting base 300 has connection ports 304, one end of which is connected to the liquid supply conduit 501 via a pipe fitting 302, and the other end of which is connected to the sample addition needle 400 via a sample addition needle adapter 301. There are four connection ports 304, which are arranged at equal intervals inside the vibration mounting base 300. The pump tube clamp base 303 is configured to clamp the liquid supply conduit 501 in order to hold the liquid supply conduit 501 vertically.

[0148] The drive controller 600 is asymmetrical with respect to the vibration drive circuit 601. motion The system includes a 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 vibrating member 100 and drives the vibrating mounting base 300 to perform asymmetric reciprocating motion according to the asymmetric reciprocating motion control program 602. The drive controller 600, using the position signal acquisition module 604, collects real-time position feedback signals from the position sensor 103 of the vibrating member 100, compares them in real time with the asymmetric reciprocating motion control program, and feeds them back to the position correction module 603, thereby adjusting the control parameters of the vibration drive circuit 601 and achieving closed-loop control of asymmetric reciprocating motion.

[0149] The drive controller 600 is further electrically connected to the first dynamic positioning member 200, the second dynamic positioning member 900, and the fluid drive device 500, and provides power to each of them.

[0150] The method for producing microdroplets using the above-described control device includes the following steps. a) The sample addition needle adapter is moved to the sponge, the syringe pump drives the liquid suction conduit to draw up the carrier liquid, the electric two-position three-way switching valve is switched, the syringe pump discharges the carrier liquid from the sample addition needle adapter, the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve and sample addition needle adapter are filled with carrier liquid and free of air bubbles, and the sponge removes the excess carrier liquid discharged from the lower end opening of the sample addition needle adapter. The carrier liquid is a second liquid that is incompatible with the sample liquid, and the carrier liquid is an oil mainly composed of hydrocarbons, silicon or fluorocarbons, such as mineral oil, tetradecane, or FC40. b) Insert the sample addition needle into the sample addition needle adapter filled with carrier liquid, and connect the sample addition needle to the sample addition needle adapter. Here, the liquid storage volume of the sample addition needle is 60 μL, the material used to make the sample addition needle 400 is polypropylene (PP, with a contact angle of 88° in pure aqueous solution), the inner diameter of the liquid discharge opening of the sample addition needle 400 is 120 μm, and the outer diameter is 400 μm. c) Move the sample dosing needle to the sponge, switch the electric two-position three-way switching valve, and repeat step a) to fill the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve, sample dosing needle adapter, and sample dosing needle with carrier liquid and ensure there are no air bubbles, and remove any excess carrier liquid discharged from the opening of the sample dosing needle with the sponge. d) Move the sample-adding needle from step c) above the liquid level in the first open container containing the first liquid, and move it downward so that the liquid outlet of the sample-adding needle comes into contact with and is immersed in the aqueous solution (first liquid). Switch the electric two-position three-way switching valve and repeat step a) to draw 20 μL of the first liquid into the sample-adding needle. e) Move the sample-adding needle from step d) out of the first opening container and move it above the second opening container containing the mineral oil (second liquid), and move it downward so that the liquid outlet of the sample-adding needle comes into contact with and is immersed in the second liquid, with the immersion depth of the sample-adding needle in the liquid surface of the second liquid being 0.3 mm. f) The first liquid in the sample injection needle is discharged from the liquid outlet of the sample injection needle under the drive of the syringe pump, and the drive controller 600 is used as a drive signal generator to periodically reciprocate the sample injection needle asymmetrically below or across the liquid surface of the second liquid, so that the vibration motor in the microdroplet generation mechanism drives the sample injection needle adapter to cause the liquid discharge opening of the sample injection needle to reciprocate asymmetrically, and the position-time waveform of the motion is a combination of a square wave and a sawtooth wave as shown in Figure 11, 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. A 1 nL microdroplet is prepared, and the microimaging of the microdroplet is as shown in Figure 14, the volume of the droplet is 1 nL, and the CV value of the volume is 2.1%.

[0151] Comparative Example 1 Comparative Example 1 differs from Example 1 in that the microdroplet generation mechanism vibrates symmetrically along the central axis of the vibration output rod, the vibration waveform is a sine wave (shown in Figure 12a), the vibration width is 1.2 mm, the vibration frequency is 100 Hz, the microsyringe flow rate is 100 nL / s, and the injection volume is 20 μL. The other structures, including the microdroplet generation mechanism, and the conditions for the microdroplet production method are the same as in Example 1. The produced microdroplets are shown in Figure 15, and the volume of the droplets is non-uniform, with a volume CV value of 35%. Further high-speed microscopic imaging observation revealed that droplet cleavage occurred in both of the two half-periods of vibration, and that, due to defects such as burrs caused by processing of the sample-adding needle, in some cases the droplet at the liquid discharge opening of the sample-adding needle was not cleaved by the oil phase shear force in one period, making it easy to generate droplets of 1 nL, 1.5 nL, or 2 nL.

[0152] Comparative Example 2 Comparative Example 2 differs from Example 1 in that the microdroplet generation mechanism vibrates symmetrically along the central axis of the vibration output rod, the vibration waveform is a square wave (shown in Figure 12b), the amplitude is 1.2 mm, the vibration frequency is 200 Hz, the flow rate of the microsyringe is 200 nL / s, and the injection volume is 20 μL. The other structures, including the microdroplet generation mechanism, and the conditions for the microdroplet production method are the same as in Example 1. The produced microdroplets are shown in Figure 16, and the CV value of the droplet size is 15%. Further high-speed microscopic imaging observation revealed that within one vibration period, the liquid discharge opening of the sample docking needle generates one droplet each in the upper and lower half-cycles. However, due to defects such as axial misalignment caused by processing of the sample docking needle, the volume of the droplet generated in the upper half-cycle and the droplet generated in the lower half-cycle do not match, resulting in a large volume error.

[0153] Comparative Example 3 Comparative Example 3 differs from Example 1 in that the microdroplet generation mechanism does not include the connecting guide structure 105. That is, vibration The output rod 104 is directly connected to the vibration mounting base 300 via the connecting member 106. The rest of the structure is the same as in Example 1. Microdroplets were fabricated using the same microdroplet fabrication method as in Example 1. The vibration noise of the vibration mechanism was large, and resonance was significant. The CV value of the fabricated microdroplets was 7.5%, and the uniformity of the microdroplet volume was worse than in Example 1, resulting in a large volume error. When attaching the sample dosing needle, there is no connecting guide structure, so the vibrating member cannot resist the torque and radial forces. After 34 uses, the connection point between the galvanometer mirror vibration output rod and the vibration motor bent, making it impossible to insert the sample dosing needle properly.

[0154] Comparative Example 4 Comparative Example 4 differs from Example 2 in that the microdroplet generation mechanism oscillates symmetrically around the central axis of the vibration output rod 104, while the other structures, including the microdroplet generation mechanism, and the conditions for the microdroplet production method are the same as in Example 2. The specific parameters for the symmetrical reciprocating motion are that the vibration waveform is a square wave (Figure 12b), the vibration width is 1.2 mm, the vibration frequency is 100 Hz, the flow rate of the microsyringe is 100 nL / s, and the injection volume is 20 μL. The uniformity of the produced microdroplets was poor, and the CV value of the microdroplet volume was 8.5%, indicating a large volume error.

[0155] Comparative Example 5 Comparative Example 5 differs from Example 2 in that the microdroplet generation mechanism does not include the connecting guide structure 105. That is, vibration The output rod 104 is directly connected to the vibration mounting base 300 via the connecting member 106. The other structures are the same as in Example 2. Microdroplets were fabricated using the same microdroplet fabrication method as in Example 2. The fabricated microdroplets are shown in Figure 12a. The specific parameters for the symmetrical reciprocating motion are a sinusoidal vibration waveform, a vibration width of 1.2 mm, a vibration frequency of 100 Hz, a microsyringe flow rate of 100 nL / s, and an injection volume of 20 μL. The uniformity of the fabricated microdroplets was poor, and the CV value of the microdroplet volume was 7.4%, indicating a large volume error. The vibration noise of the vibration mechanism was high, and resonance was significant. When attaching the sample dosing needle, there was no connecting guide structure, so the vibrating member could not resist the torque and radial forces. After 25 uses, the galvanometer mirror vibration output rod bent, making it impossible to insert the sample dosing needle normally.

[0156] Comparative Example 6 Comparative Example 6 differs from Example 2 in that the microdroplet generation mechanism does not include the sample addition needle adapter 301. That is, the liquid supply conduit 501 passes through the vibration mounting base 300 to the sample addition needle 4 00It is directly connected to the . The other structures are the same as in Example 2. Microdroplets were prepared using the same microdroplet preparation method as in Example 2. The prepared microdroplets are shown in Figure 12a. The specific parameters for the symmetrical reciprocating motion are a sinusoidal vibration waveform, a vibration width of 1.2 mm, a vibration frequency of 100 Hz, a microsyringe flow rate of 100 nL / s, and an injection volume of 20 μL. The uniformity of the prepared microdroplets was poor, and the CV value of the microdroplet volume was 30%, resulting in a large volume error. When attaching the sample addition needle, there is no sample addition needle adapter, so the sealing is not good, and leakage tends to make the droplet generation volume inaccurate.

[0157] Table 1 shows the parameters of the vibrating members and the droplet generation results for each of the above embodiments and comparative examples.

[0158] [Table 1]

[0159] As can be seen from the above embodiments, by employing the asymmetric reciprocating motion provided by this application, it is possible to effectively avoid non-uniformity in droplet size caused by asymmetry due to processing defects in the sample dosing needle and injection molding errors in the opening.

[0160] Examples 3-6 Examples 3-6 show that the first dynamic positioning member moves the sample addition needle to 0.5 mm, 1.0 mm, 1.5 mm, and 2.5 mm below the liquid surface. mm This differs from Example 1 in that the droplets are positioned at a specific depth. After droplets are generated by the asymmetric vibration microdroplet generation mechanism and stacked flat at the bottom of the container, they were observed and imaged with a microscope, and the results were as follows. Example 3: The sample addition needle was positioned 0.5 mm below the liquid surface, and the resulting droplet is shown in Figure 22. The resulting droplet was uniform and had a volume of 1 nL. Example 4: The sample addition needle was positioned 1.0 mm below the liquid surface, and the resulting droplet is shown in Figure 23. The resulting droplet was uniform and had a volume of 1 nL. Example 5: The sample addition needle was positioned 1.5 mm below the liquid surface, and the resulting droplet is shown in Figure 24. The resulting droplet was uniform and had a volume of 1 nL. Example 6: The sample addition needle was positioned 2.5 mm below the liquid surface, and the resulting droplet is shown in Figure 25. done Although the droplets were uniform, they were disturbed by the oil phase and some were fragmented.

[0161] As can be seen from the experiment, when the sample addition needle is positioned 0.0 to 1.5 mm below the liquid surface, the generated droplets are uniform, have accurate volume, and can settle regularly during the droplet generation process without interference from the sample addition needle.

[0162] As can be seen from the experimental results of Examples 3 to 6, when using the control device for microdroplet production provided in this application, it is necessary to combine an asymmetric vibrating microdroplet production mechanism with a first dynamic positioning member in order to effectively control the insertion depth of the sample dosing needle into the liquid surface and to ensure uniformity of droplet size and the stability and reliability of the droplet production system. In the actual use of the droplet production device, droplets are usually produced in an array container using an array of sample dosing needles. Liquid level errors due to processing errors, assembly errors of the multiple sample dosing needles, and volume errors of the oil phase in the container are all unavoidable. Furthermore, as the number of droplets produced increases, the liquid level of the second liquid gradually rises, and the insertion depth of the sample dosing needle into the liquid surface gradually falls outside the acceptable depth range. The first dynamic positioning member with a dynamic height adjustment function can ensure that the above errors and liquid level changes during the droplet production process do not affect the stability and reliability of the system.

[0163] Examples 7-18 Examples 7-18 differ from Example 1 in that the vibration frequency is fixed at 120 Hz, and the sample flow rates of the first liquid 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. Under the above conditions, droplets were generated by the asymmetric vibration microdroplet generation mechanism, and after the generated droplets were piled flat at the bottom of the container, they were observed by imaging with a microscope. As shown in Figure 26, the volumes of the generated droplets were 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 size is uniform, and the volume of the droplet is equal to the flow velocity divided by the vibration frequency.

[0164] Examples 7 to 18 verified the advantages of the controllable droplet generation of the control device for microdroplet production provided by the present invention.

[0165] Examples 19-28 Examples 19-28 differ from Example 1 in the sample flow rate of the first liquid and the vibration frequency of the sample addition needle, as shown in Table 2. The theoretical volume of droplets generated by asymmetric vibration using a control device for microdroplet production is obtained by dividing the flow rate by the vibration frequency. The volumes of the droplets generated in Examples 19-28 matched the actual and theoretical volumes of the generated droplets, and the linear correlation between the volume of the generated droplets and the theoretical volume value matched the theoretically calculated linear correlation (R 2 =0.9999, Figure 27), and the volumes were 200 pL, 500 pL, 1 nL, 5 nL, 10 nL, 50 nL, 100 nL, 500 nL, 1 μL, and 2 μL, respectively.

[0166] Examples 19-28 demonstrate that by adopting the present invention, size-adjustable droplet generation can be rapidly achieved by adjusting the flow rate, amplitude, and frequency, the generation conditions are flexible and controllable, and controllable generation of microdroplets with adjustable volumes of the order of 5 (200 pL to 2 μL) can be achieved, far exceeding the controllable range of droplet generation by conventional microfluidic chip methods, and without the need to change the structure of the sample dosing needle. Compared to conventional microfluidic chip droplet generation techniques, the apparatus provided by the present invention allows for direct setting of droplet size, eliminating the need to determine droplet size by observing droplet volume and optimizing parameters based on experience, and the droplet volume is not affected by changes in the inner diameter of the sample dosing needle, resulting in very good consistency.

[0167] [Table 2] TIFF0007856749000003.tif472

[0168] Example 29 This embodiment verifies the performance of multi-volume digital PCR detection using a control device for microdroplet preparation provided in this application. Conventional digital PCR systems, such as the QX200 from Bio-Rad, Thermo Fisher Scientific's QuantStudio3D, and Stilla's Naica, have a constant volume of generated droplets or microreactions, with droplet volume ranging from 0.5 to 0.8 nL. The dynamic linear detection range is typically 10 5 This is an order of magnitude. The dynamic range is narrow, and there is a trade-off between sensitivity and quantization limit. This embodiment uses a control device for microdroplet production to generate multiple droplet arrays of predetermined volumes as needed, and further improves the accuracy and dynamic detection range of digital PCR. improveBased on a typical digital PCR Poisson distribution model, the upper limit of quantification for microdroplets of different volumes was estimated. As can be seen from the calculations, as the volume of the droplet decreases, the upper limit of quantification for the droplet increases. This is mainly because, in the same reaction system, smaller microdroplets have more divisible units, and therefore, at the same concentration, the ratio of positive droplets to the total number of droplets differs, thus improving the detection limit. Based on the theoretical model, the quantitative dynamic range for microdroplets of different volumes was theoretically calculated, and the linear detection dynamic range of digital PCR was predicted under different droplet volume conditions. The 95% confidence quantification intervals for droplets of 0.2nL, 0.5nL, 1nL, 2.5nL, and 5nL volumes are shown by the dashed lines in different forms in Figure 29. The integrated 95% confidence quantification intervals for the five different volume droplets are shown by the solid curve in Figure 29. Theoretically, the dynamic quantification range can be calculated using different volume methods. 5 10 6 It can be seen that it can be extended to the order (as shown in Table 3).

[0169] [Table 3] remarks: [1] The quantitative limit for each condition is the concentration observed when the fraction of positive droplets is 0.95. [2] The detection limit for each condition is the presence of five positive droplets in a single reaction.

[0170] The actual experimental procedure is as follows: The configured digital PCR reaction system had a volume of 25 μL and consisted of 12.5 μL of 2× dPCR Super Mix (Beijing Da Microbial Co., Ltd.), 6.9 μL of deionized water, 0.6 μL of DNA polymerase (Beijing Da Microbial Co., Ltd.), 2.5 μL of 10× PCR primer probes, and 2.5 μL of nucleic acid template. The target to be detected was the EIF5B (eukaryotic translation initiation factor 5B) gene in human genomic DNA (gDNA) (shown in Table 4).

[0171] [Table 4]

[0172] gDNA samples (TaqMan Control Genomic DNA, Applied Biosystems, USA) were sequentially diluted with 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. Similar to the other experimental conditions in Example 1, a microdroplet generation control device was used at a frequency of 120 Hz with flow rates of 24 nL / s, 60 nL / s, 120 nL / s, 300 nL / s, and 600 nL / s, respectively, to successfully generate droplets of 0.2 nL, 0.5 nL, 1 nL, 2.5 nL, and 5 nL. As a result, as shown in Figure 28, the generated droplets formed a planar monolayer droplet array on a flat-bottom well plate (Beijing Da Microbial Co., Ltd.). Flat-bottom well plates were amplified in a plate PCR amplifier (Beijing Da Microbiology Co., Ltd.) using a PCR procedure of 45 cycles at 95°C for 5 minutes, 94°C for 20 seconds, and 58°C for 1 minute, and finally held at 25°C. After amplification, the flat-bottom well plates were transferred to a fluorescence reader to collect droplet fluorescence images, and the results were analyzed using digital PCR analysis software to obtain absolute quantification results of the EIF5B gene at different concentrations and droplet volumes.

[0173] The results are shown in Figure 29. As can be seen from Figure 29, by generating microdroplets of different volumes such as 0.2nL, 0.5nL, 1nL, 2.5nL, and 5nL, it is possible to achieve 1 to 10 5 The dynamic range of detection and quantification was investigated within the range of human genome gDNA template concentrations of copies / μL. Linear regression of the quantification results for droplets of each volume revealed that the R values ​​for quantification of 0.2nL, 0.5nL, 1nL, 2.5nL, and 5nL microdroplets were 0.979, 0.980, 0.995, 0.993, and 0.987, respectively. When collaborative quantification was performed for all droplet volumes, the R value was 0.996.

[0174] The results described above demonstrate that by comprehensively statistically analyzing the quantitative results of droplets of different volumes, the dynamic range of quantitative detection in digital PCR can be significantly expanded, thereby improving the accuracy of quantification. Currently, the linear range of mainstream fluorescence quantitative PCR is 5 to 7 log10 concentration ranges, while imported digital PCR using the chip method typically has a linear range of 4 to 5 log10 concentration ranges, resulting in a narrower linear dynamic range than fluorescence quantitative PCR. The droplet generation control device provided in this application not only significantly reduces the consumable cost of digital PCR and enables automated droplet array production, but also further expands the linear dynamic range of digital PCR through multi-volume technology, which is of great significance in the widespread adoption of digital PCR applications such as quantitative detection of viral load and high-sensitivity detection of rare mutations.

[0175] Exemplary embodiments of the control device for microdroplet generation provided herein have been described and / or illustrated in detail above. However, the embodiments of this application are not limited to the specific embodiments described herein, and conversely, the components and / or steps of each embodiment may be used separately and independently of other components and / or steps described herein. Each component and / or step of one embodiment may be used in combination with other components and / or steps of other embodiments. When introducing the elements / components / etc. described and / or illustrated herein, the terms “one,” “1,” and “above,” etc., indicate that there are one or more elements / components / etc. The terms “include,” “contain,” and “have” are used to mean open inclusion and mean that additional elements / components / etc. may exist in addition to the elements / components / etc. listed. Also, the terms “first,” “second,” etc., in the claims and specification are used only as reference numerals and do not limit the numbers of the subject. [Explanation of symbols]

[0176] 1-Asymmetric vibrating microdroplet generation mechanism, 100-Vibrating member, 101-Housing, 102-Vibrator, 103-Position sensor, 104-Vibration outputRod, 105 - connecting guide structure, 1051 - spline sleeve, 1052 - spline shaft, 1053 - first bearing, 1054 - second bearing, 106 - connecting member, 120 - support base, 200 - first dynamic positioning member, 201 - positioning member lifting / displacement mechanism, 202 - liquid level detection mechanism, 300 - vibration mounting base, 301 - sample addition needle adapter, 302 - pipe fitting, 303 - pump tube clamp base 304-Connection port, 400-Sample addition needle, 401-Liquid supply opening of sample addition needle, 402-Liquid discharge section of sample addition needle, 403-Liquid discharge opening of sample addition needle, 404-Liquid storage section of sample addition needle, 500-Fluid drive device, 501-Liquid supply conduit, 502-Liquid suction conduit, 503-Micro syringe, 504-Oil storage bottle, 505-Carrier liquid, 600-Drive controller, 601-Vibration drive circuit, 602-Asymmetric motion Control program, 603-position correction module, 604-position signal acquisition module, 605-power supply control connection cable, 700-second liquid, 701-second opening container, 702-liquid level of the second liquid, 703-microdroplets of the first liquid, 800-first liquid, 801-first opening container, EP-equilibrium position of vibration, RP1 and RP2-reflection positions located on both sides of the equilibrium position, 900-second dynamic positioning member.

Claims

1. A control device for producing microdroplets, wherein the control device includes a means for producing microdroplets and a means for controlling fluid. The microdroplet production means includes an asymmetric vibrating microdroplet generation mechanism and a first dynamic positioning member, the asymmetric vibrating microdroplet generation mechanism includes a vibrating member and a sample dosing needle, and the first dynamic positioning member is fixedly connected to the asymmetric vibrating microdroplet generation mechanism. The first dynamic positioning member is configured to position the asymmetric vibrating microdroplet generation mechanism, The fluid control means includes a fluid drive device and a conduit, one end of the conduit is connected to the fluid drive device, and the other end of the conduit is connected to the asymmetric vibrating microdroplet generation mechanism, and the fluid drive device is configured to set the flow velocity in order to cause the sample addition needle to absorb and drain liquid through the conduit. The asymmetric vibrating microdroplet generation mechanism further includes a vibration mounting base, a connecting guide structure, and a drive controller, wherein the vibration mounting base has a connection port, a pipe fitting, and a sample dosing needle adapter, one end of the connection port is connected to the liquid supply conduit of the conduit via a pipe fitting, and the other end is connected to the sample dosing needle by the sample dosing needle adapter. The vibrating member includes a housing, a vibrator, and a vibrating output rod. The vibration output rod of the vibrating member is connected to the vibration mounting base via the connecting guide structure and configured to provide power to the vibration mounting base, and the drive controller is electrically connected to the vibrating member. The control device for producing microdroplets is characterized in that the asymmetric vibrating microdroplet generation mechanism drives the vibration mounting base via the drive controller to perform asymmetric reciprocating vibration or asymmetric reciprocating oscillation in a direction along the central axis of the vibration output rod, thereby causing a sample-adding needle filled with a first liquid to perform asymmetric reciprocating motion below the liquid surface of a second liquid, thereby generating microdroplets of the first liquid.

2. The control device according to claim 1, characterized in that the asymmetric vibrating microdroplet generation mechanism generates one microdroplet within one motion period, and the one motion period is the path of the vibrating member that goes from a reflection point RP1, through an equilibrium point EP to a reflection point RP2, and then returns from the reflection point RP2, again going through the equilibrium point EP to the reflection point RP1.

3. The control device according to claim 2, characterized in that the central axis of the sample dosing needle is perpendicular to the axis of the vibration output rod.

4. The control device according to claim 3, characterized in that the sample-adding needle generates one microdroplet within one period of asymmetric reciprocating vibration or asymmetric reciprocating oscillation of the vibrating mounting base.

5. The vibration frequency of the vibrating member is 10 to 1000 Hz. The control device according to claim 3, characterized in that the vibration amplitude of the vibrating member is 0.1 to 5 mm.

6. The oscillation frequency of the aforementioned vibration mounting base is 10 to 1000 Hz. The control device according to claim 3, characterized in that the distance between the liquid discharge opening of the sample dosing needle and the axis of the vibration output rod is 10 to 100 mm, and the oscillation angle range of the vibration mounting base is 0.05 to 10°.

7. The control device according to claim 3, characterized in that the vibration mounting base and the vibration output rod are connected by a coupling.

8. The vibrating member further includes a position sensor, and the drive controller achieves closed-loop control of motion by collecting real-time position feedback signals from the position sensor. The position sensor is one of the following: a grid scale sensor, a capacitive position sensor, a resistive sensor, a current sensor, or a differential transformer sensor. The control device according to claim 3, characterized in that the closed-loop control of the asymmetric reciprocating motion is performed by the drive controller, which collects a real-time position feedback signal from the position sensor of the vibrating member using a position signal acquisition module, compares it in real time with the asymmetric reciprocating motion control program, and feeds it back to the position correction module to adjust the control parameters of the vibration drive circuit.

9. The asymmetric vibrating microdroplet generation mechanism further includes a support and fixing base for fixing the vibrating member, The control device according to claim 3, wherein the asymmetric vibrating microdroplet generation mechanism further includes a pump tube clamp base for clamping the liquid supply conduit.

10. The aforementioned connection ports are multiple, and the multiple connection ports are provided at equal intervals inside the vibration mounting base. The control device according to claim 3, characterized in that the number of connection ports is 1 to 96.

11. The control device according to claim 10, wherein the connection guide structure 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.

12. The control device according to claim 10, wherein the connecting guide structure includes a first bearing and a second bearing, one end of the vibration mounting base is connected to the vibration output rod through the first bearing, the other end of the vibration mounting base is connected to the second bearing, and the first bearing is a bearing having an axial locking edge.

13. The control device according to claim 3, further comprising a sample dosing needle removal mechanism for automatically removing the sample dosing needle after microdroplets have been generated.

14. The sample dosing needle has a conical tubular structure with open ends, one end being a liquid supply opening for tight insertion with the sample dosing needle adapter, and the other end being a liquid discharge opening for generating microdroplets, the inner diameter of the liquid discharge opening being 20 to 300 μm and the outer diameter being 150 to 600 μm. The control device according to claim 3, characterized in that the liquid storage volume range of the sample addition needle is 5 to 500 μL.

15. When the vibrating mounting base performs the asymmetric reciprocating motion, the motion of the liquid dispensing portion of the sample-adding needle has one equilibrium point and two reflection points at both ends of the equilibrium point, and the position-time curve of the motion is configured to be asymmetric on both sides of either reflection point. The control device according to claim 3, characterized in that the asymmetric waveform of the periodic motion of the liquid discharge part of the sample dosing needle is at least one asymmetric combination of a sine wave, sawtooth wave, trapezoidal wave, triangular wave, and square wave.

16. The control device according to claim 3, characterized in that the vibrating member is configured as a mechanism that generates continuous or intermittent motion, and the vibrating member is one selected from an electromagnetic vibrating device, a piezoelectric ceramic vibrating device, an eccentric wheel vibrating device, a servo motor, a voice coil motor, and a galvanometer motor.

17. The first dynamic positioning member includes a positioning member lifting displacement mechanism for controlling the lifting and lowering of the asymmetric vibrating microdroplet generation mechanism. The control device according to claim 1, wherein the first dynamic positioning member further includes a liquid level detection mechanism for assisting the first dynamic positioning member in accurately positioning the opening of the sample dosing needle.

18. The control device further includes a second dynamic positioning member, which is used to fix and move the first opening container and the second opening container. The control device according to claim 17, wherein the drive controller provides power to the fluid drive device, the first dynamic positioning member and the second dynamic positioning member, the first open container contains a first liquid, the sample addition needle draws in the first liquid to generate microdroplets of the first liquid, the second open container contains a second liquid, the sample addition needle performs asymmetric reciprocating vibration or asymmetric oscillation below the liquid surface of the second liquid to discharge the first liquid from the sample addition needle to generate microdroplets of the first liquid.

19. 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 to 1000 μL. The control device according to claim 18, wherein the second opening container is a two-dimensional flat-bottom sample cell array for stacking the generated microdroplets, and the second opening container contains 24, 32, 96, or 384 flat-bottom sample cells of equal volume.

20. The control device according to claim 19, characterized in that the fluid drive device is a pulsation-free drive pump, and there is one or more fluid drive devices.

21. The control device according to claim 19, wherein the conduit includes a liquid supply conduit and a liquid suction conduit, one end of the liquid suction conduit is connected to the fluid drive device via one valve port of an electric two-position three-way switching valve, the other end of the liquid suction conduit is inserted into an oil storage device containing a carrier liquid, one end of the liquid supply conduit is connected to the fluid drive device via one valve port of an electric two-position three-way switching valve, and the other end of the liquid supply conduit is connected to the asymmetric vibrating microdroplet generation mechanism.

22. The control device according to claim 21, further comprising preparation means, the preparation means comprising a sample-adding needle rack, an oil removal mechanism, and a waste receiver, wherein the oil removal mechanism is located above the waste receiver to remove excess carrier liquid discharged from the lower end opening of the sample-adding needle adapter or the opening of the sample-adding needle, and the sample-adding needle is removed above the waste receiver.

23. Using a fluid drive device, the conduit, sample injection needle adapter, and sample injection needle are driven to fill the sample injection needle with the first liquid. A method for producing microdroplets using a control device according to any one of claims 1 to 22, comprising bringing a sample-dosing needle filled with a first liquid into contact with a second liquid, and under the drive of a fluid drive device, the asymmetric vibrating microdroplet generation mechanism drives a sample-dosing needle adapter to cause the sample-dosing needle to reciprocate asymmetrically below the liquid surface of the second liquid, thereby generating microdroplets of the first liquid.

24. Using a fluid drive device, the conduit, sample addition needle adapter, and sample addition needle are driven, and before filling the sample addition needle with the first liquid, the method further: a) The sample addition needle adapter is moved to the oil removal mechanism, the fluid drive device drives the liquid suction conduit to draw up carrier liquid from the oil reservoir, and switches the electric two-position three-way switching valve, the fluid drive device discharges the carrier liquid from the sample addition needle adapter, and the carrier liquid is filled into the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve and sample addition needle adapter, and the oil removal mechanism removes the excess carrier liquid discharged from the lower end opening of the sample addition needle adapter. b) Insert the sample addition needle into the sample addition needle adapter filled with carrier solution, and connect the sample addition needle to the sample addition needle adapter. The method according to 23, characterized in that c) the sample addition needle is moved to the oil removal mechanism, the electric two-position three-way switching valve is switched, and step a) is repeated to fill the liquid suction conduit, liquid supply conduit, electric two-position three-way switching valve, sample addition needle adapter and sample addition needle with carrier liquid and free of air bubbles, and the oil removal mechanism removes excess carrier liquid discharged from the opening of the sample addition needle.

25. The step of using a fluid drive device to drive the conduit, sample addition needle adapter, and sample addition needle, and filling the inside of the sample addition needle with the first liquid, The method according to 24, characterized in that it includes moving the sample-adding needle of step c) above the liquid surface of the first open container containing the first liquid, moving it downward, bringing the liquid outlet of the sample-adding needle into contact with and immersing it in the first liquid, switching the electric two-position three-way switching valve, and drawing the first liquid into the sample-adding needle to fill the inside of the sample-adding needle with the first liquid.

26. The method according to 25, characterized in that the volume of the first liquid drawn in from the liquid outlet of the sample-adding needle is smaller than the volume of the connection cavity of the sample-adding needle.

27. The method according to 23, further comprising removing the sample addition needle after the preparation of a first liquid microdroplet is complete, and then reinserting the sample addition needle to prepare another first liquid microdroplet.

28. The method according to 23, characterized in that the sample-adding needle performs asymmetric reciprocating vibration or asymmetric oscillation below the liquid surface of the second liquid, and the fluid drive device is used to set the flow velocity and discharge the first liquid from the sample-adding needle to generate microdroplets of the first liquid.

29. The method according to 23, characterized in that the sample-adding needle generates only one microdroplet within one period of asymmetric reciprocating motion below the liquid surface of the second liquid, and when the vibrating mounting base is in asymmetric reciprocating motion, the one-period motion of the liquid discharge portion of the sample-adding needle has one equilibrium point and two reflection points at both ends of the equilibrium point, and is configured to go from reflection point RP1 to reflection point RP2 via equilibrium point EP, and then return from reflection point RP2 to reflection point RP1 again via equilibrium point EP.

30. The method according to 23, characterized in that the first dynamic positioning member drives an asymmetric vibrating microdroplet generation mechanism to position the sample dosing needle below the liquid surface of the second liquid, and during the asymmetric vibrating process, the average depth of the sample dosing needle inserted into the liquid surface is dynamically maintained within a range of 0 to 2.0 mm below the liquid surface.

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