Microfluidic device and droplet monitoring system

Abstract

The utility model provides a kind of microfluidic device and droplet monitoring system, the negative pressure extraction device of microfluidic device generates liquid drop in liquid storage container through liquid inlet negative pressure extraction to droplet storage cavity, then, sample liquid in sample container is extracted to droplet storage cavity through sample inlet negative pressure, so that sample liquid and liquid drop generating liquid are mixed and generate target liquid drop. The utility model shortens monitoring cycle, synchronously improves monitoring sensitivity and specificity, reduces monitoring cost.

Description

Technical Field

[0001] This utility model belongs to the field of microbial monitoring technology, and in particular relates to a microfluidic device and a droplet monitoring system. Background Technology

[0002] Biopharmaceuticals are drugs produced using organisms (such as bacteria, yeast, animal cells, or plant cells) or their components (such as organelles and enzymes) for the prevention, treatment, and diagnosis of diseases. Current technologies for microbial monitoring of biopharmaceuticals often suffer from the following problems: Firstly, the monitoring cycle is lengthy, making it difficult to meet the needs of immediate quality control in drug production and urgent clinical diagnosis. Furthermore, false negative results are easily caused by unmet growth conditions for specific microorganisms or mutual inhibition between microorganisms. Secondly, while rapid monitoring technologies are fast, they face challenges in balancing sensitivity and specificity. Immunological monitoring is prone to cross-reactivity leading to false positives, and improving specificity often sacrifices sensitivity. Simultaneously, the accuracy of monitoring results can be affected by the sample matrix. Summary of the Invention

[0003] This invention addresses the technical problems of existing microbial monitoring technologies, such as long monitoring cycles and the difficulty in balancing sensitivity and specificity, by providing a microfluidic device and a droplet monitoring system.

[0004] In view of the above technical problems, this utility model provides a microfluidic device, including: The microfluidic chip has a liquid inlet, a sample inlet, a liquid outlet, and a droplet storage cavity. The droplet storage cavity has a cavity inlet and a cavity outlet at opposite ends. The cavity inlet is connected to the liquid inlet and the sample inlet, and the cavity outlet is connected to the liquid outlet. Liquid storage container, connected to the liquid inlet; The sample container is connected to the sample inlet; A negative pressure extraction device is connected to the liquid outlet; the negative pressure extraction device is used to extract the droplet generating liquid in the liquid storage container into the droplet storage cavity through the liquid inlet under negative pressure, and then extract the sample liquid in the sample container into the droplet storage cavity through the sample inlet under negative pressure, so that the sample liquid and the droplet generating liquid are mixed to generate the target droplet.

[0005] Optionally, the microfluidic chip is further provided with a sample channel and an inlet channel connecting the cavity inlet and the liquid inlet; one end of the sample channel is connected to the sample inlet, and the other end is connected to a preset intersection point of the inlet channel.

[0006] Optionally, the sample flow channel is provided with a curved section.

[0007] Optionally, the liquid inlet channel includes a first channel, a second channel, and a third channel; the first channel and the second channel are both connected between the preset junction point and the liquid inlet, and the third channel is connected between the cavity inlet and the preset junction point; the first channel and the second channel are arranged on opposite sides of the sample channel.

[0008] Optionally, the microfluidic chip is further provided with a liquid outlet channel, which connects the cavity outlet and the liquid outlet.

[0009] Optionally, the negative pressure extraction device is an injection device.

[0010] Optionally, the liquid storage container includes a liquid storage pool with a first opening at the top, and a first breathable membrane covering the first opening; and / or The sample container includes a sample pool with a second opening at the top, and a second breathable membrane covering the second opening.

[0011] Optionally, the microfluidic chip includes a channel layer and a substrate layer, the liquid inlet, the sample inlet and the liquid outlet are all disposed on the channel layer, and the droplet storage cavity is formed between the channel layer and the substrate layer.

[0012] This invention also provides a droplet monitoring system, including a controller and at least one of the aforementioned microfluidic devices; the controller is communicatively connected to the negative pressure extraction device.

[0013] Optionally, a transparent window is provided on the microfluidic chip at a position opposite to the droplet storage cavity, and the transparent window covers the droplet storage cavity; the droplet monitoring system further includes an optical device communicatively connected to the controller, the optical device being positioned opposite to the droplet storage cavity to monitor the target droplet in the droplet storage cavity through the transparent window.

[0014] In this invention, the microfluidic device utilizes target droplet microfluidic technology to precisely control the generation of target droplets. Specifically, firstly, a droplet-generating liquid in the storage container is drawn into the droplet storage chamber through a negative pressure extraction device via an inlet. Then, sample liquid in the sample container is drawn into the droplet storage chamber through a negative pressure inlet, allowing the sample liquid and droplet-generating liquid to mix and generate target droplets. This process utilizes two immiscible liquids (sample liquid and droplet-generating liquid) to form tiny target droplets in the droplet storage chamber of the microfluidic device. Thus, the microfluidic device can discretize the sample liquid, dividing it into tiny target droplets. Each target droplet can then function as an independent reaction unit. The sample liquid then reacts and is monitored within nanoliter or even picoliter droplets, effectively preventing sample dilution. This allows the microorganisms to be analyzed (such as individual bacteria or fungi) to be analyzed in a relatively high concentration environment, resulting in a more complete and efficient interaction between the microorganisms and the monitoring reagents, thereby improving monitoring sensitivity. Furthermore, this invention integrates functions such as rapid droplet preparation, droplet capture, droplet incubation, and real-time microbial monitoring into a single microfluidic chip. Since the entire monitoring process involves no droplet transfer, it effectively reduces false negative results caused by sample loss and exhibits high specificity. Moreover, the aforementioned target droplet microfluidic technology offers advantages such as no cross-contamination, rapid reaction, simple device, good repeatability, and ease of precise control. In the field of biopharmaceutical microbial monitoring, target droplet microfluidic technology can precisely control the size, shape, and composition of droplets, providing a stable and uniform monitoring environment for microorganisms, thus improving the repeatability and reliability of monitoring. Because target droplet microfluidic technology monitors within tiny droplets, the required reagent and sample volumes are significantly reduced, lowering monitoring costs.

[0015] Secondly, the microfluidic device adopts a negative pressure extraction mode directly connected to the negative pressure extraction device, which can quickly generate a large number of target droplets with uniform particle size. If there are microorganisms in the sample solution in these independent target droplets, the contact area and reaction efficiency between the microorganisms in the sample solution and the culture medium or monitoring reagent will be improved, thereby making the concentration of microorganisms in a single droplet relatively higher, which greatly improves the monitoring sensitivity and speed.

[0016] Finally, microfluidic devices offer advantages such as small droplet preparation volume, convenient operation, low manufacturing cost, and ease of modular design. Furthermore, their miniaturization and integration reduce the use of large instruments and the space required in laboratories, further lowering monitoring costs and resource consumption. Moreover, they do not require complex laboratory facilities or a large number of professional personnel for operation, enabling timely monitoring of microorganisms in sample solutions even in resource-scarce scenarios (such as remote medical stations, field emergency services, and small community clinics). Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Figure 1 This is a schematic diagram of the structure of a microfluidic device provided in an embodiment of the present invention.

[0019] Figure 2 This is a partial structural schematic diagram of a microfluidic device provided in an embodiment of the present invention.

[0020] Figure 3 This is a schematic diagram of the structure of the microfluidic chip of the microfluidic device provided in one embodiment of the present invention.

[0021] Figure 4 This is a partial structural schematic diagram of a droplet monitoring system provided in one embodiment of the present invention.

[0022] Figure 5 This is a flowchart of a droplet monitoring method provided in an embodiment of the present invention.

[0023] Figure 6 This is a real-time droplet image of the target droplet formed in one embodiment of the present invention; Figure 7 This is a real-time droplet image corresponding to Escherichia coli after incubation in the target droplet; Figure 8 This is a real-time droplet image of Escherichia coli after incubation in a target droplet using the zirconia staining method.

[0024] The reference numerals in the accompanying drawings are as follows: 10. Microfluidic chip; 101. Liquid inlet; 102. Sample inlet; 103. Liquid outlet; 104. Droplet storage chamber; 1041. Chamber inlet; 1042. Chamber outlet; 105. Sample channel; 1051. Bend section; 106. Liquid inlet channel; 1061. First channel; 1062. Second channel; 1063. Third channel; 107. Preset confluence point; 108. Liquid outlet channel; 109. Channel layer; 110. Substrate layer; 20. Liquid storage container; 201. Liquid storage tank; 202. First breathable membrane; 30. Sample container; 301. Sample tank; 302. Second breathable membrane; 40. Negative pressure extraction device; 50. Optical equipment; 60. Connector. Detailed Implementation

[0025] To make the technical problems solved, technical solutions, and beneficial effects of this utility model clearer, the present utility model will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this utility model and are not intended to limit this utility model.

[0026] like Figures 1 to 4 As shown, this embodiment of the present invention provides a microfluidic device, comprising: The microfluidic chip 10 is provided with an inlet 101, an inlet 102, an outlet 103 and a droplet storage cavity 104. The droplet storage cavity 104 is provided with a cavity inlet 1041 and a cavity outlet 1042 at opposite ends. The cavity inlet 1041 is connected to the inlet 101 and the inlet 102, and the cavity outlet 1042 is connected to the outlet 103.

[0027] A liquid storage container 20 is connected to the liquid inlet 101. Further, the liquid storage container 20 includes a liquid storage pool 201 with a first opening at the top, and a first breathable membrane 202 covering the first opening. The first breathable membrane 202 can be adhered to the edge of the first opening to achieve the effect of the liquid storage container 20 being breathable but waterproof. The first breathable membrane 202 can be a waterproof breathable membrane or a hydrophobic breathable membrane, etc., which are breathable but waterproof films. Specifically, the waterproof breathable membrane can be made of polytetrafluoroethylene, thermoplastic polyurethane rubber, or polyurethane, etc. The waterproof breathable membrane allows gaseous water molecules (water vapor) to pass through, but blocks liquid water penetration, achieving the effect of "breathable but waterproof." The hydrophobic breathable membrane can be made of polyvinylidene fluoride, polypropylene, or hydrophobically modified materials, etc. The hydrophobic breathable membrane repels liquid water through the hydrophobicity of its surface while maintaining gas permeability.

[0028] The sample container 30 is connected to the sample inlet 102. Further, the sample container 30 includes a sample pool 301 with a second opening at the top, and a second breathable membrane 302 covering the second opening. The second breathable membrane 302 can be adhered to the edge of the second opening to achieve the effect of the sample container 30 being breathable but waterproof. The second breathable membrane 302 can be a waterproof or hydrophobic breathable membrane, or other breathable but waterproof thin film. Preferably, the first breathable membrane 202 and the second breathable membrane 302 can be breathable membranes with pores having a pore size of 0.2 μm. In this case, the 0.2 μm pore size allows the breathable membrane to filter out most microorganisms and larger particles while maintaining good gas permeability, thereby achieving sterile and pollution-free gas exchange.

[0029] A negative pressure extraction device 40 is connected to the outlet 103. The negative pressure extraction device 40 is used to draw the droplet-generating liquid from the storage container 20 into the droplet storage chamber 104 under negative pressure through the inlet 101, and then draw the sample liquid from the sample container 30 into the droplet storage chamber 104 under negative pressure through the inlet 102, so that the sample liquid and the droplet-generating liquid are mixed to generate the target droplets. Further, the negative pressure extraction device 40 is an injection device. This injection device can also be an injection pump, thus enabling fully automated droplet preparation operations through automatic control of the injection pump.

[0030] In a further embodiment, the negative pressure extraction device 40 needs to draw the droplet-generating liquid from the liquid storage container 20 through the inlet 101 under negative pressure until it fills the droplet storage cavity 104, and then draw the sample liquid from the sample container 30 through the inlet 102 under negative pressure into the droplet storage cavity 104, so that the sample liquid and the droplet-generating liquid mix and generate target droplets that cover the droplet storage cavity 104. Thus, after the droplet-generating liquid fills the droplet storage cavity 104, the sample liquid will enter the droplet-generating liquid and mix with it. Therefore, the mixing will be more uniform and easier to generate target droplets. Furthermore, after the sample liquid and the droplet-generating liquid mix and generate target droplets that cover the droplet storage cavity 104, it will be easier to monitor the target droplets in the droplet storage cavity 104 to obtain the microbial monitoring results of the sample liquid.

[0031] Understandably, the components of the droplet generating liquid and the sample liquid can be set according to requirements, as long as they enable the droplet generating liquid to mix with the sample liquid and generate the target droplet. For example, the sample liquid can refer to a liquid containing or not containing microorganisms, and may also contain colorimetric agents such as resazurin indicator; while the droplet generating liquid can be droplet generating oil, wherein the oil phase of the droplet generating oil is the continuous phase, and the aqueous phase of the sample liquid is the dispersed phase, thereby forming an oil-in-water droplet, i.e., the target droplet, in the droplet storage cavity 104.

[0032] In this invention, the liquid storage container 20, the sample container 30, and the negative pressure extraction device 40 can all be detachably mounted on the microfluidic chip 10 via connectors 60. Connector 60 can be a Luer female connector, in which case a Luer male connector is provided at the lower end of the liquid storage container 20, the sample container 30, and the negative pressure extraction device 40. Preferably, the materials used for the liquid storage tank 201, the sample tank 301, the flow channel layer 109, the base layer 110, the Luer female connector, and the Luer male connector include, but are not limited to, polymethyl methacrylate, polycarbonate, cyclic olefin copolymers, and cyclic olefin polymers.

[0033] Furthermore, the microfluidic chip 10 includes a channel layer 109 and a substrate layer 110. The inlet 101, the sample inlet 102, and the outlet 103 are all disposed on the channel layer 109, and the droplet storage cavity 104 is formed between the channel layer 109 and the substrate layer 110. When the microfluidic chip 10 needs to be assembled, a thermoforming bonding machine is used to bond the channel layer 109 and the substrate layer 110. The inlet 101, the sample inlet 102, the outlet 103, and the droplet storage cavity 104 are formed on the channel layer 109 in the form of grooves or holes. Therefore, after the channel layer 109 and the substrate layer 110 are thermoformed and bonded, a closed microchannel is formed. Next, connect the connector 60 to the liquid inlet 101, sample inlet 102, and liquid outlet 103 on the flow channel layer 109 to complete the assembly. Then, seal the assembled part in an aluminum foil bag for later use.

[0034] In this embodiment, when a microfluidic device is needed, the microfluidic chip 10 is first taken out of the aluminum foil bag, and the liquid storage container 20, sample container 30 and negative pressure extraction device 40 are respectively connected to the liquid inlet 101, sample inlet 102 and liquid outlet 103 on the microfluidic chip 10 through the connector 60. Next, an appropriate amount of droplet-generating liquid is added to the storage container 20 using a pipette. Then, the negative pressure extraction device 40 is controlled to enter the negative pressure extraction mode. At this time, the pressure inside the negative pressure extraction device 40 decreases, thereby creating a negative pressure in the flow channel connecting the negative pressure extraction device 40 and the storage container 20 (i.e., the flow channel between the inlet 101 and the outlet 103). This negative pressure causes the droplet-generating liquid in the storage container 20 to be drawn into the droplet storage chamber 104 through the inlet 101 and finally flow to the outlet 103. At this point, it can be confirmed that the droplet-generating liquid fills the droplet storage chamber 104, and the negative pressure extraction device 40 can be controlled to exit the negative pressure extraction mode, stopping the extraction of droplet-generating liquid. In this process, filling the droplet storage chamber 104 with droplet-generating liquid in advance ensures that the target droplets can be generated stably and that the size and shape of the target droplets are controllable.

[0035] Next, use a pipette to add an appropriate amount of sample solution to the sample container 30, and then use the first breathable membrane 202 and the second breathable membrane 302 with adhesive backing to seal the first opening of the liquid storage container 20 and the second opening of the sample container 30, respectively. Then, the negative pressure extraction device 40 is controlled to enter the negative pressure extraction mode. At this time, the pressure inside the negative pressure extraction device 40 is reduced, thereby forming a negative pressure in the flow channel connecting the negative pressure extraction device 40 and the sample container 30 (that is, the flow channel between the inlet 102 and the outlet 103). The negative pressure causes the sample liquid in the sample container 30 to be drawn in through the inlet 102. Before entering the droplet storage cavity 104, it begins to merge with the droplet generating liquid and quickly generates a target droplet with uniform particle size. After the target droplet with uniform particle size enters the droplet storage cavity 104, it quickly spreads evenly throughout the droplet storage cavity 104. Then, the negative pressure extraction device 40 is controlled to exit the negative pressure extraction mode and stop extracting the sample liquid. At this time, the target droplet stops moving and remains stationary in the droplet storage cavity 104. During the process described above, as the target droplets gradually stack towards the outlet 103, the method to determine whether the target droplets have completely filled the droplet storage cavity 104 can be as follows: when the optical device 50 observes and confirms that the target droplets flow out of the outlet 103 (or enter the outlet channel mentioned later), it means that the target droplets have completely filled the droplet storage cavity 104. At this point, the negative pressure extraction device 40 can be controlled to exit the negative pressure extraction mode. Afterwards, the microorganisms in the sample liquid within the target droplets can be incubated in the droplet storage cavity 104. Simultaneously, the optical device 50, such as an inverted biological microscope or a lensless microscope, can be used to observe the color changes of the target droplets and the morphological changes of the droplet contents in the droplet storage cavity 104 in real time, thereby determining the microbial monitoring results of the sample liquid.

[0036] In this invention, the microfluidic device utilizes target droplet microfluidic technology to precisely control the generation of target droplets. Specifically, firstly, the droplet-generating liquid in the storage container 20 is drawn into the droplet storage cavity 104 through the inlet 101 by the negative pressure extraction device 40. Then, the sample liquid in the sample container 30 is drawn into the droplet storage cavity 104 through the inlet 102, so that the sample liquid and the droplet-generating liquid are mixed to generate target droplets that cover the droplet storage cavity 104. The above process utilizes two immiscible liquids (sample liquid and droplet-generating liquid) to form tiny target droplets in the droplet storage cavity 104 of the microfluidic device. Thus, the microfluidic device can discretize the sample solution, breaking it down into tiny target droplets. Each droplet can then function as an independent reaction unit. The sample solution reacts and is monitored within nanoliter or even picolinate droplets, effectively preventing sample dilution. This allows for the analysis of microorganisms (such as individual bacteria or fungi) in a relatively high concentration environment, resulting in a more thorough and efficient interaction between the microorganisms and the monitoring reagents, thereby improving monitoring sensitivity. Furthermore, this invention integrates rapid droplet preparation, droplet capture, droplet incubation, and real-time microbial monitoring into a single microfluidic chip 10. Since there is no droplet transfer operation during the entire monitoring process, false negative results due to sample loss are effectively reduced, resulting in high specificity. Moreover, the aforementioned target droplet microfluidic technology offers advantages such as no cross-contamination, rapid reaction, simple device, good repeatability, and easy precise control. In the field of microbial monitoring for biopharmaceuticals, targeted droplet microfluidics can precisely control the size, shape, and composition of droplets, providing a stable and uniform monitoring environment for microorganisms and helping to improve the repeatability and reliability of monitoring. Because targeted droplet microfluidics monitors within tiny droplets, the required reagent and sample volumes are significantly reduced, thus lowering monitoring costs.

[0037] Secondly, the microfluidic device employs a direct-connect negative pressure extraction mode, enabling the rapid generation of numerous uniform target droplets (thousands per second), significantly improving sample processing speed and efficiency, and achieving high-throughput microbial monitoring. This efficient droplet generation capability allows for the processing of large numbers of samples in a short time, greatly enhancing monitoring efficiency and throughput. Furthermore, each of these independent target droplets acts as an independent culture unit, capable of encapsulating down to a single microbial cell (such as bacteria or fungi), providing a relatively closed enrichment environment for microorganisms. The discrete target droplets also concentrate the monitoring signal, reduce background noise, and make weak monitoring signals easier to identify, thereby improving monitoring sensitivity. Thus, when microorganisms are present in the sample solution, the contact area and reaction efficiency between the microorganisms in the target droplet and the culture medium or monitoring reagent are increased, resulting in a relatively higher concentration of microbial samples within a single droplet, significantly improving monitoring sensitivity and speed. These target droplets provide a relatively independent growth environment for individual microbial cells, enabling them to grow and reproduce without competition or inhibition from other microorganisms, which helps in the discovery of rare and slow-growing microorganisms in complex samples.

[0038] Finally, microfluidic devices offer advantages such as small droplet preparation volume, convenient operation, low manufacturing cost, and ease of modular design. Furthermore, their miniaturization and integration reduce the use of large instruments and the space required in laboratories, further lowering monitoring costs and resource consumption. Moreover, they do not require complex laboratory facilities or a large number of professional personnel for operation, enabling timely monitoring of microorganisms in sample solutions even in resource-scarce scenarios (such as remote medical stations, field emergency services, and small community clinics).

[0039] In some embodiments, such as Figure 2 and Figure 3As shown, the microfluidic chip 10 is also provided with a sample channel 105 and an inlet channel 106 connecting the cavity inlet 1041 and the liquid inlet 101; one end of the sample channel 105 is connected to the sample inlet 102, and the other end is connected to the preset confluence point 107 of the inlet channel 106. That is, at this time, the droplet generating liquid will sequentially enter the droplet storage cavity 104 through the liquid inlet 101, the inlet channel 106, and the cavity inlet 1041; while the sample liquid will sequentially enter the droplet storage cavity 104 through the sample inlet 102, the sample channel 105, the preset confluence point 107, the inlet channel 106 between the preset confluence point 107 and the cavity inlet 1041, and the cavity inlet 1041. The droplet generating liquid and the sample liquid enter the channel containing a common flow channel (the inlet flow channel 106 between the preset confluence point 107 and the cavity inlet 1041). Thus, after the droplet generating liquid fills the droplet storage cavity 104, the common flow channel is also filled with droplet generating liquid. Then, when the sample liquid in the sample container 30 is drawn in through the inlet 102 by negative pressure, it can start to merge with the droplet generating liquid in the common flow channel before entering the droplet storage cavity 104, and quickly generate target droplets with uniform particle size here. Then, after the target droplets with uniform particle size enter the droplet storage cavity 104, they quickly spread evenly throughout the entire droplet storage cavity 104.

[0040] Furthermore, the microfluidic chip 10 is also provided with a liquid outlet channel 108, which connects the cavity outlet 1042 and the liquid outlet 103. That is, a liquid outlet channel 108 is also provided between the liquid outlet 103 and the droplet storage cavity 104. When the droplet generating liquid is drawn in, it can be observed whether the droplet generating liquid fills the entire droplet storage cavity 104. That is, when the droplet generating liquid enters the liquid outlet channel 108 or flows out from the liquid outlet 103, it can be considered that the droplet generating liquid has filled the entire droplet storage cavity 104. At the same time, the liquid outlet channel 108 can also be used to observe whether the target droplet is generated and fills the entire droplet storage cavity 104 when the sample liquid is drawn in. That is, when the target droplet enters the liquid outlet channel 108 or flows out from the liquid outlet 103, it can be considered that the target droplet has filled the entire droplet storage cavity 104.

[0041] Furthermore, such as Figure 3 As shown, the sample flow channel 105 is provided with a curved section 1051; the design of the curved section 1051 on the sample flow channel 105 can make the liquid flow more stable. The curved section 1051 ensures the stability of the dispersed phase flow by balancing the flow velocity distribution, reducing eddies and turbulence, improving fluid dynamic stability, and mitigating flow impact, thereby improving working efficiency and controllability.

[0042] Furthermore, such as Figure 3As shown, the liquid inlet channel 106 includes a first channel 1061, a second channel 1062, and a third channel 1063. The first channel 1061 and the second channel 1062 are both connected between the preset intersection point 107 and the liquid inlet 101, and the third channel 1063 is connected between the cavity inlet 1041 and the preset intersection point 107. The first channel 1061 and the second channel 1062 are arranged on opposite sides of the sample channel 105. In this embodiment, the first channel 1061, the second channel 1062, the third channel 1063, and the sample channel 105 intersect at the preset intersection point 107, thereby forming a "cross-shaped" channel at the preset intersection point 107. The target droplet is then produced at the "cross-shaped" channel and quickly spreads out in the droplet storage cavity 104.

[0043] like Figure 4 As shown, this embodiment of the invention also provides a droplet monitoring system, including a controller and at least one of the aforementioned microfluidic devices; the controller is communicatively connected to the negative pressure extraction device 40. The microfluidic chips 10 in the microfluidic devices can be combined to form a multi-channel microfluidic chip 10, i.e., as shown... Figure 4 As shown, when facing the need for monitoring multiple samples and high throughput, multiple microfluidic chips 10 can be connected in parallel within the same droplet monitoring system (i.e., multiple microfluidic devices can be combined) to achieve synchronous processing by multiple microfluidic chips 10. By increasing the number of parallel channels, not only can the monitoring throughput be increased, but multiple samples can also be processed simultaneously, enabling microbial monitoring of multiple samples and meeting the requirements for rapid release of sterile products. The aforementioned multi-channel design allows the droplet monitoring system to monitor multiple samples at once, further improving the efficiency and flexibility of monitoring, and making it suitable for large-scale microbial monitoring needs. The droplet monitoring system in this invention has a good modular design foundation, and functional modules can be flexibly combined and expanded according to different needs, further enhancing the practicality and flexibility of the droplet monitoring system.

[0044] Understandably, the droplet monitoring system of this invention is used to execute the following droplet monitoring method. The various devices used in the droplet monitoring method, such as pipettes, can all be considered part of the droplet monitoring system, and will not be described in detail here. Each module in the controller can be implemented entirely or partially through software, hardware, or a combination thereof. These modules can be embedded in the controller in hardware form or independent of the controller, or stored in the controller in software form, so that the controller can call and execute the operations corresponding to each module.

[0045] In some embodiments, such as Figures 1 to 4As shown, a transparent window is provided on the microfluidic chip 10 at a position opposite to the droplet storage cavity 104, and the transparent window covers the droplet storage cavity 104; the droplet monitoring system also includes an optical device 50 communicatively connected to the controller, the optical device 50 being positioned opposite to the droplet storage cavity 104 for monitoring target droplets in the droplet storage cavity 104 through the transparent window. Wherein, as Figure 1 As shown, the imaging lens of the optical device 50 is positioned parallel to the droplet storage cavity 104 directly below it, and can observe or capture real-time images of the droplets in the droplet storage cavity 104 through a transparent window. Preferably, the optical device 50 can be an inverted biological microscope or a lensless microscope, etc. The optical device 50 enables real-time monitoring of the microbial growth within the target droplet during the incubation process at the single-cell level, thereby achieving rapid detection of low-abundance microorganisms.

[0046] like Figure 5 As shown, this utility model embodiment also provides a droplet monitoring method, applied to the controller of the droplet monitoring system, the droplet monitoring method comprising: S100: Receive the first negative pressure extraction signal and control the negative pressure extraction device 40 to extract the droplet generating liquid in the liquid storage container 20 into the droplet storage cavity 104 through the inlet 101 under negative pressure. Specifically, after the microfluidic device is assembled, an appropriate amount of droplet generating liquid can be added to the liquid storage container 20 using a pipette. Then, the first negative pressure extraction signal can be triggered by button triggering, voice triggering, or other methods on the human-machine interface. After receiving the first negative pressure extraction signal, the controller will control the negative pressure extraction device 40 to enter the negative pressure extraction mode. At this time, the pressure inside the negative pressure extraction device 40 decreases, thereby forming a negative pressure in the flow channel connecting the negative pressure extraction device 40 and the liquid storage container 20 (i.e., the flow channel between the inlet 101 and the outlet 103). Then, the droplet generating liquid in the liquid storage container 20 is sucked into the droplet storage cavity 104 through the inlet 101 by the negative pressure. Preferably, step S200 can be performed after the droplet generating liquid is drawn into the droplet storage chamber 104 in step S100 to fill the droplet storage chamber 104. Understandably, in step S100, when the droplet generating liquid is drawn in, if it is determined that the droplet generating liquid flows out of the outlet 103 or enters the outlet channel, it means that the droplet generating liquid has filled the entire droplet storage chamber 104. Therefore, the negative pressure extraction device 40 can be controlled to exit the negative pressure extraction mode and stop extracting the droplet generating liquid.

[0047] S200: Upon receiving the second negative pressure extraction signal, the sample liquid containing microorganisms in the sample container 30 is extracted under negative pressure through the inlet 102 into the droplet storage chamber 104, so that the sample liquid mixes with the droplet generating liquid and generates the target droplet. Understandably, in some embodiments, the second negative pressure extraction signal can be automatically generated after the droplet generating liquid fills the droplet storage chamber 104. Alternatively, the second negative pressure extraction signal can be triggered by button presses or voice commands on the human-machine interface. After receiving the second negative pressure extraction signal, the controller will control the negative pressure extraction device 40 to enter the negative pressure extraction mode. The pressure inside the negative pressure extraction device 40 decreases, thereby creating a negative pressure in the flow channel connecting the negative pressure extraction device 40 and the sample container 30 (i.e., the flow channel between the inlet 102 and the outlet 103). The negative pressure causes the sample liquid in the sample container 30 to be drawn in through the inlet 102, and then begins to merge with the droplet generating liquid in the common flow channel before entering the droplet storage cavity 104 (the inlet flow channel 106 between the preset confluence point 107 and the cavity inlet 1041 of the droplet storage cavity 104). At this point, a target droplet with uniform particle size is rapidly generated, and then the target droplet with uniform particle size enters the droplet storage cavity 104. Understandably, after the target droplet enters the droplet storage cavity 104, it will quickly spread out in the droplet storage cavity 104. Preferably, after the target droplet has spread out, the negative pressure extraction device 40 can be controlled to exit the negative pressure extraction mode and stop extracting the sample liquid. At this time, the target droplet stops moving and remains stationary in the droplet storage cavity 104, which makes it easier to monitor it in step S300.

[0048] S300. Monitor the target droplet within the droplet storage cavity 104 to obtain the microbial monitoring results of the sample solution. Understandably, the sample solution in this invention may or may not contain microorganisms. The microbial monitoring results determined by the droplet monitoring method and system in this invention can indicate whether microorganisms are present in the sample solution. In this step, the target droplet begins to incubate in the droplet storage cavity 104. Furthermore, the microbial monitoring results of the sample solution can be determined by monitoring the color changes of the target droplet and the morphological changes of its contents within the droplet storage cavity 104. For example, if the sample solution contains microorganisms, the microorganisms will grow synchronously during the incubation process of the target droplet. At this time, the incubation status of the microorganisms can be determined jointly based on the color changes of all target droplets and the morphological changes of their contents. Therefore, the microbial monitoring result can be determined as the presence of microorganisms in the sample solution (positive result). Further, when microorganisms are present in the sample solution, the microbial monitoring results can not only characterize the presence of microorganisms but also include information indicating whether the incubation status of the microorganisms is normal. When the sample solution does not contain microorganisms, the color and contents of the target droplet will not change because there is no microbial incubation and growth. In this case, the microbial monitoring result can be determined to be that there are no microorganisms in the sample solution (negative result).

[0049] The microfluidic device in the above embodiments of this invention utilizes target droplet microfluidic technology to precisely control the generation of target droplets. Specifically, firstly, the droplet-generating liquid in the storage container 20 is drawn into the droplet storage cavity 104 through the inlet 101 by the negative pressure extraction device 40. Then, the sample liquid in the sample container 30 is drawn into the droplet storage cavity 104 through the inlet 102, so that the sample liquid and the droplet-generating liquid are mixed to generate target droplets that fill the droplet storage cavity 104. The above process utilizes two immiscible liquids (sample liquid and droplet-generating liquid) to form tiny target droplets in the droplet storage cavity 104 of the microfluidic device. Thus, the microfluidic device can discretize the sample solution, breaking it down into tiny target droplets. Each droplet can then function as an independent reaction unit. The sample solution reacts and is monitored within nanoliter or even picolinate droplets, effectively preventing sample dilution. This allows for the analysis of microorganisms (such as individual bacteria or fungi) in a relatively high concentration environment, resulting in a more thorough and efficient interaction between the microorganisms and the monitoring reagents, thereby improving monitoring sensitivity. Furthermore, this invention integrates rapid droplet preparation, droplet capture, droplet incubation, and real-time microbial monitoring into a single microfluidic chip 10. Since there is no droplet transfer operation during the entire monitoring process, false negative results due to sample loss are effectively reduced, resulting in high specificity. Moreover, the aforementioned target droplet microfluidic technology offers advantages such as no cross-contamination, rapid reaction, simple device, good repeatability, and easy precise control. In the field of microbial monitoring for biopharmaceuticals, targeted droplet microfluidics can precisely control the size, shape, and composition of droplets, providing a stable and uniform monitoring environment for microorganisms and helping to improve the repeatability and reliability of monitoring. Because targeted droplet microfluidics monitors within tiny droplets, the required reagent and sample volumes are significantly reduced, thus lowering monitoring costs.

[0050] Secondly, the microfluidic device employs a direct-connect negative pressure extraction mode, enabling the rapid generation of numerous uniform target droplets (thousands per second), significantly improving sample processing speed and efficiency, and achieving high-throughput microbial monitoring. This efficient droplet generation capability allows for the processing of large numbers of samples in a short time, greatly enhancing monitoring efficiency and throughput. Furthermore, each of these independent target droplets acts as an independent culture unit, capable of encapsulating down to a single microbial cell (such as bacteria or fungi), providing a relatively closed enrichment environment for microorganisms. The discrete target droplets also concentrate the monitoring signal, reduce background noise, and make weak monitoring signals easier to identify, thereby improving monitoring sensitivity. Thus, when microorganisms are present in the sample solution, the contact area and reaction efficiency between the microorganisms in the target droplet and the culture medium or monitoring reagent are increased, resulting in a relatively higher concentration of microbial samples within a single droplet, significantly improving monitoring sensitivity and speed. These target droplets provide a relatively independent growth environment for individual microbial cells, enabling them to grow and reproduce without competition or inhibition from other microorganisms, which helps in the discovery of rare and slow-growing microorganisms in complex samples.

[0051] In some embodiments, the droplet monitoring system further includes an optical device 50 communicatively connected to the controller; the imaging lens of the optical device 50 is positioned parallel to the droplet storage cavity 104 directly below it, and can observe or capture real-time droplet images in the droplet storage cavity 104 through a transparent window on the microfluidic chip 10. Preferably, the optical device 50 may be an inverted biological microscope or a lensless microscope, etc.

[0052] Further, in step S300, monitoring the target droplet within the droplet storage cavity 104 to obtain the microbial monitoring results of the sample solution includes: Real-time droplet images of the target droplet within the droplet storage cavity 104 are acquired using the optical device 50; wherein, for example... Figure 6 As shown, the real-time droplet image is the actual image of the target droplet in the droplet storage cavity 104 at the current moment, captured by the optical device 50.

[0053] The real-time droplet image is segmented and standardized to obtain multiple standard images. Specifically, the acquired real-time droplet image is segmented to a fixed size. For example, when the acquired image size is 1024×1024, it can be segmented into multiple sub-images with sizes of 32×32 or 64×64. Then, the segmented sub-images can be scaled to match the image size (e.g., 25×25, 50×50, etc.) required by the preset image recognition model, thus obtaining the standard image. Understandably, when the sub-image size matches the image size required by the preset image recognition model, there is no need to scale the sub-image further; the sub-image can be directly determined as the standard image.

[0054] The target droplets in all the standard images are color-recognized using a preset image recognition model to obtain the color recognition results corresponding to the real-time droplet images. Understandably, in this embodiment, the sample solution includes chromogenic agents such as resazurin indicator. Thus, when microorganisms are present in the sample solution, the color of the target droplets will undergo specific color changes under the influence of the chromogenic agent at each stage of microbial incubation. For example, referring to… Figure 8 As shown, the azurlan indicator will initially appear purple, then gradually change to pink and white at different incubation stages. Thus, when microorganisms are present in the sample solution, the target droplet will undergo different color changes at each incubation stage corresponding to the microorganisms (corresponding to different standard color change information). Therefore, by performing color recognition on standard images, and then determining the color recognition result corresponding to the real-time droplet image based on the individual recognition results of all standard images, the color recognition result can characterize whether the target droplet's color has undergone a preset color change. For example, as... Figure 8 As shown, if the individual identification result corresponding to some target droplets remains unchanged as purple, while the individual identification result corresponding to other target droplets changes from purple to pink, and the individual identification result corresponding to other target droplets changes from pink to white, then the color identification result can be that some target droplets change from purple to pink (first incubation stage 4.5-5h), and then change from pink to white (second incubation stage 5-5.5h).

[0055] The microbial monitoring result is determined based on the color recognition result, which is used to characterize whether the sample solution contains microorganisms. That is, since the actual incubation time of the target droplet in the droplet storage cavity 104 is fixed, the incubation stage to which the current actual incubation time belongs can be determined (if the actual incubation time falls within the incubation time range corresponding to a certain incubation stage, then it is determined that the current incubation stage is the one to which it belongs). Then, it is determined whether the current color recognition result matches the standard color change information corresponding to that incubation stage. If they match, the microbial monitoring result indicates the presence of microorganisms in the sample solution, and the microbial monitoring result can include information that the current incubation status of the microorganism is normal. If they do not match, and the color of the target droplet undergoes a color change different from the standard color change information, the microbial monitoring result indicates the presence of microorganisms in the sample solution, and the microbial monitoring result can include information that the incubation status of the microorganism is abnormal (e.g., the presence of different types of microorganisms affecting the color change, or poor microbial growth). If they do not match, and the color of the target droplet does not change, the microbial monitoring result indicates that no microorganisms are present in the sample solution.

[0056] In some embodiments, the step of performing color recognition on all the standard images using a preset image recognition model to obtain color recognition results corresponding to each of the standard images includes: The pigment features in each standard image are extracted by a preset image recognition model, and a one-dimensional color vector in each standard image is determined based on the pigment features. In this step, the preset image recognition model can perform color recognition based on the SVM recognition method of the three primary colors of red, green and blue. That is, the preset image recognition model extracts the pigment features of the entire standard image, normalizes the pixel values ​​corresponding to the pigment features to the range of [0, 255], and flattens the normalized pixel value data into a one-dimensional color vector (e.g., if the pixel values ​​corresponding to the pigment features are 25, 25 and 3 respectively, then the one-dimensional color vector is 25×25×3=1875 dimensions).

[0057] The color one-dimensional vectors corresponding to all the standard images are averaged to obtain the color recognition result corresponding to the real-time droplet image. Understandably, in this embodiment, the color one-dimensional vectors of all standard images are aggregated, and then their average value is taken; this average value is then determined as the color recognition result corresponding to the real-time droplet image. In this embodiment, each incubation stage corresponds to a standard one-dimensional vector range. When the color recognition result (i.e., the average of all color one-dimensional vectors) falls within the aforementioned standard one-dimensional vector range, it is confirmed that the current color recognition result matches the standard color change information corresponding to that incubation stage; conversely, when the color recognition result exceeds the standard one-dimensional vector range, it is confirmed that the current color recognition result does not match the standard color change information corresponding to that incubation stage.

[0058] Furthermore, before determining the microbial monitoring result based on the color recognition result, the method further includes: All the aforementioned standard images are subjected to black-and-white normalization processing to obtain a black-and-white image corresponding to each standard image. Understandably, in the next step, the wall thickness of the target droplet needs to be identified using a preset thickness recognition model. In this embodiment, the standard images cannot be expanded or eroded because color variations exist in the standard images, and the wall thickness of the target droplet will change with time and color (e.g., ...). Figure 7 As shown, black agglomerates will be generated in the target droplet. Such agglomerates may adhere to the wall of the target droplet, which will cause changes in the wall thickness. In this embodiment, the colors in the standard image are normalized to the range of [0, 1], so that the standard image contains only black and white colors, so as to highlight the wall thickness lines of the target droplet and make its thickness more obvious.

[0059] The wall thickness of the target droplets in all the black and white images is identified using a preset thickness recognition model to obtain wall thickness information corresponding to each black and white image. Based on this wall thickness information, the wall thickness level corresponding to the real-time droplet image is determined. In this embodiment, the preset thickness recognition model is trained using a convolutional neural network (CNN) to learn line thickness features. Specifically, the CNN can learn wall thickness features based on samples of manually labeled wall thickness data. Once the trained CNN can recognize and output pixel-level wall thickness information, it is identified as the preset thickness recognition model. At this point, the preset thickness recognition model is used to identify the wall thickness of the target droplets in all the black and white images, outputting wall thickness information corresponding to each black and white image. The controller can then determine the wall thickness level corresponding to the real-time droplet image based on this wall thickness information. Alternatively, once the trained CNN can recognize and output the wall thickness level corresponding to the pixel-level wall thickness information, it is identified as the preset thickness recognition model. At this point, the wall thickness of the target droplets in all the black and white images is identified using a preset thickness recognition model. After the preset thickness recognition model outputs the identified wall thickness information, the wall thickness level corresponding to the real-time droplet image can be directly determined and output based on all the wall thickness information. The controller does not need to further determine the wall thickness level.

[0060] In the above embodiments, determining the wall thickness level corresponding to the real-time droplet image based on all the wall thickness information can be achieved by dividing the wall thickness level into multiple target levels (e.g., three-level classification: fine, medium, and coarse, or five-level classification). Each target level corresponds to a wall thickness range. Each wall thickness information is matched with all wall thickness ranges, and the ratio of all wall thickness information belonging to the same wall thickness range is calculated. The target level corresponding to the wall thickness range with the highest ratio is determined as the wall thickness level corresponding to the real-time droplet image.

[0061] The step of determining the microbial monitoring result based on the color recognition result includes: The microbial monitoring result is determined based on the color recognition result and the wall thickness level. That is, in this embodiment, each incubation stage corresponds to a standard wall thickness level. Understandably, if the standard wall thickness level matches the standard wall thickness level corresponding to the current incubation stage, and the color recognition result matches the standard color change information corresponding to that incubation stage, then the microbial monitoring result indicates the presence of microorganisms in the sample solution, and the microbial monitoring result may include information that the current incubation status of the microorganism is normal. If the color recognition result does not match the standard color change information corresponding to that incubation stage, and the color of the target droplet does not change, and the standard wall thickness level does not match the standard wall thickness level corresponding to the current incubation stage, then the microbial monitoring result indicates the absence of microorganisms in the sample solution.

[0062] Furthermore, if the color recognition result does not match the standard color change information corresponding to the incubation stage, the color of the target droplet changes to a different color than the standard color change information, and the standard wall thickness level does not match the standard wall thickness level corresponding to the current incubation stage, then the microbial monitoring result indicates that microorganisms are present in the sample solution, and the microbial monitoring result may include information on the abnormal incubation status of the microorganism (e.g., the presence of different types of microorganisms affecting color changes, or poor microbial growth status, etc.).

[0063] In one embodiment, after monitoring the target droplet within the droplet storage cavity 104 to obtain the microbial monitoring results of the sample solution in step S300, the process includes: The microfluidic device is disposed of in a pre-designated biochemical waste bin. Understandably, disposing of the microfluidic device in the pre-designated biochemical waste bin ensures that the waste is disposed of safely and in compliance with regulations.

[0064] After monitoring is completed, the microfluidic chip 10, negative pressure extraction device 40, liquid storage container 20 and sample container 30 are disposed of together in the biochemical waste bin.

[0065] The following embodiments illustrate the solution in detail: Example 1: The droplet monitoring method of this invention was used to observe the changes in the growth status of Escherichia coli in droplets in real time. The sample solution in sample container 30 contained an appropriate amount of Escherichia coli.

[0066] The growth status of Escherichia coli in droplets was observed in real time using optical equipment 50 (inverted biological microscope), such as... Figure 7 As shown, after incubating Escherichia coli in droplets for 2.5 hours, Escherichia coli grew rapidly and clusters were visible in the center of the target droplets. At the same time, black aggregates were found to be generated in each target droplet.

[0067] Example 2: Based on the resazurin colorimetric method, the droplet monitoring method of this invention is used to observe in real time the color change of the target droplet and the growth status of Escherichia coli in the target droplet. The sample solution in the sample container 30 contains an appropriate amount of Escherichia coli and resazurin indicator mixture.

[0068] The color changes of the target droplet and the growth status of Escherichia coli within the target droplet were observed in real time using an inverted biological microscope, such as... Figure 8 As shown, with the increase of incubation time, the color of some target droplets containing Escherichia coli changed significantly (purple → pink → white), and Escherichia coli could be seen agglomerating in the center of the target droplets and forming black aggregates.

[0069] After Escherichia coli was incubated in the target droplet for 2.5 hours, the color of the target droplet changed from purple (rezatha) to pink (halogen). The change from rezatha to halogen was caused by the pH change within the target droplet. As the number of Escherichia coli gradually increased, its metabolism caused the pH within the target droplet to decrease, and the color of rezatha changed from purple (alkaline) to pink (acidic) like halogen.

[0070] After incubating Escherichia coli in the target droplet for 5 hours, a white target droplet appeared (dihydrohalothrin; halothrin was converted to dihydrohalothrin, and as the number of Escherichia coli continued to increase, reducing metabolites were produced, reducing halothrin to dihydrohalothrin, causing the target droplet to turn white). After incubating Escherichia coli in the target droplet for 5.5 hours, the number of white target droplets gradually increased, and different colored target droplets (purple, pink, and white) could be seen simultaneously in one field of view.

[0071] After incubating Escherichia coli in the target droplet for 4.5 hours, Escherichia coli grew rapidly and clumps of Escherichia coli were visible in the center of the target droplet; after incubating Escherichia coli in the target droplet for 5.5 hours, black aggregates were found to form in some of the target droplets.

[0072] Furthermore, the droplet monitoring system also includes an optical device 50 that is communicatively connected to the controller; the microbial monitoring results include the number of microorganisms and the concentration of microorganisms. Step S300, namely, monitoring the target droplet within the droplet storage cavity 104 to obtain the microbial monitoring results of the sample solution, includes: Real-time droplet images of the target droplet within the droplet storage cavity 104 are acquired using the optical device 50. The total number of target droplets and the statistical number of target droplets without microorganisms are determined based on the real-time droplet images. The number and concentration of microorganisms in the sample solution are determined based on the total quantity and the statistical quantity.

[0073] In this embodiment, the target droplets in the incubated microfluidic device can be monitored to determine the number and concentration of microorganisms in the original sample solution injected into the sample container 30 (understandably, when the sample solution does not contain microorganisms, both the number and concentration of microorganisms are 0). That is, firstly, image recognition is performed on the target droplets in the real-time droplet image to determine the total number of target droplets, and simultaneously, it is determined whether the target droplets contain microorganisms, thus determining the statistical number of target droplets without microorganisms. For example, if the total number of target droplets is N, the statistical number of target droplets containing zero microorganisms (i.e., without microorganisms) is N0.

[0074] Next, the number and concentration of microorganisms in the sample solution are determined based on the total number and the statistical count. Specifically, the proportion of target droplets without microorganisms is p0 = N0 / N. According to the Poisson distribution, p0 = e^(-λ), and this proportion can be used to calculate λ = -lnp0. Assuming the total number of target droplets is N = 20000, and the number of target droplets without microorganisms is N0 = 19000, then p0 = 0.95, and the average number of microorganisms in each target droplet is λ ≈ 0.0513. At this point, if the total volume of the original sample solution is V, and the volume of each discretized target droplet is v, since λ ≈ 0.0513, the number X and concentration of microorganisms in the original sample solution can be calculated using the Poisson distribution and the target droplet statistics. Specifically, the number of microorganisms in the sample solution X = λV / v. If the sample volume is V=100μL and the volume of each target droplet is v=1nL, then since λ≈0.0513, the number of microorganisms in the original sample is 5130, and the corresponding microbial concentration is 51300 CFU / mL.

[0075] It should be clarified that in some embodiments of this invention, the microbial monitoring results can characterize whether the sample solution contains microorganisms. In other embodiments of this invention, the microbial monitoring results may include the number and concentration of microorganisms. In still other embodiments of this invention, the microbial monitoring results can characterize both whether the sample solution contains microorganisms and the number and concentration of microorganisms, which is not limited here. That is, in this invention, while determining the microbial monitoring results characterizing whether the sample solution contains microorganisms by performing color recognition (and wall thickness recognition) on real-time droplet images, it is also possible to identify the number and concentration of microorganisms based on real-time droplet images. The two methods can be performed simultaneously without conflict.

[0076] It should be understood that the sequence number of each step in the above embodiments does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of this utility model embodiment.

[0077] The above are merely embodiments of the microfluidic device and droplet monitoring system of this utility model, and are not intended to limit this utility model. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this utility model should be included within the protection scope of this utility model.

Claims

1. A microfluidic device, characterized in that, include: The microfluidic chip has a liquid inlet, a sample inlet, a liquid outlet, and a droplet storage cavity. The droplet storage cavity has a cavity inlet and a cavity outlet at opposite ends. The cavity inlet is connected to the liquid inlet and the sample inlet, and the cavity outlet is connected to the liquid outlet. Liquid storage container, connected to the liquid inlet; The sample container is connected to the sample inlet; A negative pressure extraction device is connected to the liquid outlet; the negative pressure extraction device is used to extract the droplet generating liquid in the liquid storage container into the droplet storage cavity through the liquid inlet under negative pressure, and then extract the sample liquid in the sample container into the droplet storage cavity through the sample inlet under negative pressure, so that the sample liquid and the droplet generating liquid are mixed to generate the target droplet.

2. The microfluidic device according to claim 1, characterized in that, The microfluidic chip is also provided with a sample channel and an inlet channel connecting the cavity inlet and the liquid inlet; one end of the sample channel is connected to the sample inlet, and the other end is connected to a preset intersection point of the inlet channel.

3. The microfluidic device according to claim 2, characterized in that, The sample flow channel has a curved section.

4. The microfluidic device according to claim 2, characterized in that, The liquid inlet channel includes a first channel, a second channel, and a third channel; the first channel and the second channel are both connected between the preset junction point and the liquid inlet, and the third channel is connected between the cavity inlet and the preset junction point; the first channel and the second channel are arranged on opposite sides of the sample channel.

5. The microfluidic device according to claim 1, characterized in that, The microfluidic chip is also provided with a liquid outlet channel, which connects the cavity outlet and the liquid outlet.

6. The microfluidic device according to claim 1, characterized in that, The negative pressure extraction device is an injection device.

7. The microfluidic device according to claim 1, characterized in that, The liquid storage container includes a liquid storage pool body with a first opening at the top, and a first breathable membrane covering the first opening; and / or The sample container includes a sample pool with a second opening at the top, and a second breathable membrane covering the second opening.

8. The microfluidic device according to claim 1, characterized in that, The microfluidic chip includes a channel layer and a substrate layer. The liquid inlet, the sample inlet, and the liquid outlet are all disposed on the channel layer, and the droplet storage cavity is formed between the channel layer and the substrate layer.

9. A droplet monitoring system, characterized in that, It includes a controller and at least one microfluidic device as described in any one of claims 1 to 8; the controller is communicatively connected to the negative pressure extraction device.

10. The droplet monitoring system according to claim 9, characterized in that, The microfluidic chip has a transparent window positioned opposite the droplet storage cavity, and the transparent window covers the droplet storage cavity; the droplet monitoring system also includes an optical device communicatively connected to the controller, and the optical device is positioned opposite the droplet storage cavity to monitor the target droplet in the droplet storage cavity through the transparent window.

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