Droplet fusion system

Abstract

The application provides a droplet fusion system, comprising: a microfluidic chip; a gas supply assembly; a first laser detection unit for emitting a first detection laser, receiving a first feedback light formed after passing through a first laser detection area, and generating a first laser detection signal; a second laser detection unit for emitting a second detection laser, receiving a second feedback light formed after passing through a second laser detection area, and generating a second laser detection signal; an image acquisition unit for shooting an image of an image acquisition area; and a controller for receiving the detection signals of the first laser detection unit and the second laser detection unit and the image information of the image acquisition unit, and controlling the gas supply assembly to adjust the speed and pressure of liquid flow in the first microfluidic channel and the second microfluidic channel. The droplet fusion system of the application uses the laser detection unit and the image acquisition unit to monitor the droplet generation and fusion process, and the controller adjusts the parameters for real-time quality control, which significantly improves the reliability and efficiency of single-cell sequencing technology.

Description

Technical Field

[0001] The present application relates to the field of single-cell sequencing technology, and in particular to a droplet fusion system. Background Art

[0002] As an important bioanalytical tool, droplet microfluidics has been widely used in single-cell analysis, digital PCR, immunoassays, and other fields. By encapsulating individual cells or reaction components in tiny oily droplets, droplet technology offers unprecedented possibilities for high-throughput parallel processing. However, with the deepening of life science research and increasing application demands, simple droplet encapsulation technology has gradually revealed its limitations and is unable to meet the increasingly complex experimental needs.

[0003] For example, the development of modern biotechnology requires not only encapsulating samples in droplets for simple, independent reactions, but also more complex operations such as fusion, separation, content exchange, and multi-step reaction chains between droplets. These operations require precise manipulation within the droplets to achieve more complex and sophisticated experimental designs.

[0004] For example, large-scale biological analysis and drug screening require not only high throughput but also the exchange and mixing of contents between multiple droplets to simulate complex biological reaction environments. This demand places higher demands on droplet technology, requiring the ability to process and fuse large numbers of droplets with different components in a short period of time while ensuring consistency and reliability of each operation.

[0005] In response to the growing demand for these applications, droplet fusion technology has emerged. By fusing the contents of different droplets together, droplet fusion enables more complex chemical reactions, biological analyses, and material synthesis. This technology not only significantly enhances experimental flexibility and precision but also enables the integration of multiple different experiments on a single platform, significantly expanding the application scope of droplet microfluidics.

[0006] On the one hand, droplet fusion technology allows researchers to fuse samples and reagents in different droplets together to achieve multi-step reactions or multi-sample interactions. This capability greatly improves the complexity and flexibility of the experiment, allowing reactions that originally needed to be performed step by step to be completed continuously within the droplet. On the other hand, by performing multiple reaction steps in a single droplet, droplet fusion technology can significantly reduce the operating time and reagent consumption of the experiment, which not only improves experimental efficiency but also reduces costs, which is especially important for large-scale high-throughput screening experiments. On the other hand, droplet fusion technology can ensure the accuracy and controllability of each fusion operation, thereby improving the accuracy of the analysis results, which is particularly important in gene editing, single-molecule detection, and complex sample analysis that require fine regulation.

[0007] Although droplet fusion technology shows great application potential, it still faces many challenges in practical application:

[0008] (1) Unstable fusion success rate: Instability in the droplet fusion process remains a major problem. If the fusion efficiency is low or incomplete, it will lead to sample loss and data deviation, affecting the reliability of the experimental results.

[0009] (2) Lack of real-time monitoring methods: Currently, the success of droplet fusion often relies on post-analysis, and there is a lack of effective real-time monitoring methods to ensure the success of the fusion process. In this case, errors in the experiment are difficult to detect and correct in time, increasing the failure rate of the experiment. Summary of the Invention

[0010] In order to solve the existing technical problems, the present application provides a droplet fusion system to achieve real-time monitoring of the droplet generation frequency and the electrofusion process.

[0011] To achieve the above objectives, the technical solution of the embodiment of the present application is implemented as follows:

[0012] The present invention provides a droplet fusion system, comprising:

[0013] A microfluidic chip comprising a first microchannel, a second microchannel, and a fused microchannel, wherein the first microchannel and the second microchannel merge into the fused microchannel, a first laser detection area and a second detection area are formed on the first microchannel and the second microchannel, respectively, and an image acquisition area is formed at the intersection of the first microchannel, the second microchannel, and the fused microchannel;

[0014] A gas supply component, used for injecting gas into the first microchannel and the second microchannel;

[0015] a first laser detection unit, configured to emit a first detection laser, receive a first feedback light formed after passing through the first laser detection area, and generate a first laser detection signal;

[0016] a second laser detection unit, configured to emit a second detection laser, receive a second feedback light formed after passing through the second laser detection area, and generate a second laser detection signal;

[0017] An image acquisition unit, configured to capture an image of the image acquisition area;

[0018] a controller that receives detection signals from the first laser detection unit and the second laser detection unit and image information from the image acquisition unit, and controls the gas supply assembly to adjust the speed and pressure of the liquid flow in the first microchannel and the second microchannel based on the first laser detection signal, the second laser detection signal, and the image information.

[0019] In one embodiment, it further includes:

[0020] The third laser detection unit is used to emit a third laser signal, receive third feedback light formed after passing through the third laser detection area on the fusion microchannel, and generate a third laser detection signal.

[0021] In one embodiment, it further includes:

[0022] The fluorescence detection unit is used to emit fluorescence excitation light, receive the fluorescence excited after passing through the fluorescence detection area on the fusion microchannel, and generate a fluorescence detection signal.

[0023] In one embodiment, the first laser detection unit includes a first infrared laser emitter and a first infrared receiver; the second laser detection unit includes a second infrared laser emitter and a second infrared receiver; the third laser detection unit includes a third infrared laser emitter and a third infrared receiver; the image acquisition unit includes a camera device, and the camera device is below the image acquisition area; the fluorescence detection unit includes a fluorescence excitation light source and a fluorescence receiving module;

[0024] Among them, the first infrared laser emitter, the second infrared laser emitter, the third infrared laser emitter and the fluorescence excitation light source are all arranged above the microfluidic chip, and the first infrared receiver, the second infrared receiver, the third infrared receiver and the fluorescence receiving module are all arranged below the microfluidic chip.

[0025] In one embodiment, the image acquisition unit further includes a first adjustment mechanism, a second adjustment mechanism and a third adjustment mechanism, the camera device is installed at the movable end of the first adjustment mechanism, the first adjustment mechanism is installed at the movable end of the second adjustment mechanism, the second adjustment mechanism is installed at the movable end of the third adjustment mechanism, and the moving directions of the movable end of the first adjustment mechanism, the movable end of the second adjustment mechanism and the movable end of the third adjustment mechanism are perpendicular to each other.

[0026] In one embodiment, a first reflective element is provided above the first laser detection area and the second laser detection area, and the first infrared laser emitter and the second infrared laser emitter are relatively arranged on both sides of the first reflective element in the front-to-back direction, and the first reflective element reflects the first detection laser and the second detection laser downward.

[0027] In one embodiment, the third infrared laser emitter is disposed directly above the third laser detection area.

[0028] In one embodiment, a dichroic mirror is provided between the image acquisition area and the camera device, and the dichroic mirror transmits white light and reflects the first feedback light, the second feedback light, and the third feedback light directed downwards forward.

[0029] In one embodiment, the third infrared receiver is arranged in the front-to-back direction and directly facing the dichroic mirror, a second reflective element is arranged in front of the dichroic mirror, and the first infrared receiver and the second infrared receiver are respectively arranged on the left and right sides of the second reflective element.

[0030] In one embodiment, the fluorescence excitation light source is disposed directly above the fluorescence detection area, and the fluorescence receiving module is aligned with the fluorescence excitation light source in a vertical direction.

[0031] The droplet fusion system of the present application has at least the following beneficial effects: in the droplet fusion system of the present application, a laser detection unit and an image acquisition unit are used to achieve quality control during the droplet generation and fusion process. The laser detection unit is very sensitive to changes in refractive index and has the advantage of fast response speed, so that it can accurately detect the refractive index changes inside the droplet, thereby accurately identifying the droplet generation frequency and internal structure data; the image acquisition unit can intuitively detect the shape, size and fusion status of the droplet through image analysis, and can monitor the physical state of the droplet and the morphological changes after fusion in real time; the monitoring data of the laser detection unit and the monitoring data of the image acquisition unit can ensure the accuracy of the detection through cross-validation data, and the physical information such as the size and speed of the droplet can be calibrated with each other through joint detection to ensure the accuracy of the droplet physical property data, so that abnormal conditions in the droplet generation and fusion process can be more accurately judged; thereby, the droplet generation and fusion process can be accurately monitored in real time, and the parameters can be adjusted by the controller to perform real-time quality control, significantly improving the reliability and efficiency of single-cell sequencing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A schematic diagram of the flow direction of droplets in a microfluidic chip of a droplet fusion system according to an embodiment of the present application;

[0033] Figure 2 for Figure 1 Schematic diagram of droplet fusion in a microfluidic chip;

[0034] Figure 3 This is a schematic diagram of the framework structure of a droplet fusion system according to an embodiment of the present application;

[0035] Figure 4 for Figure 3 Schematic diagram of the structure of the droplet fusion system on the microfluidic chip;

[0036] Figure 5This is a schematic diagram of the three-dimensional structure of a droplet fusion system according to an embodiment of the present application;

[0037] Figure 6 for Figure 5 The top view of the droplet fusion system after removing the three adjustment mechanisms of the image acquisition system;

[0038] Figure 7 for Figure 6 Schematic diagram of the left side.

[0039] The components in the figure are numbered as follows:

[0040] A first laser detection unit 100 (including a first infrared laser emitter 110 and a first infrared receiver 120);

[0041] A second laser detection unit 200 (including a second infrared laser emitter 210 and a second infrared receiver 220);

[0042] a third laser detection unit 300 (including a third infrared laser emitter 310 and a third infrared receiver 320);

[0043] Image acquisition unit 400 (including camera device 410, first adjustment mechanism 420, second adjustment mechanism 430, and third adjustment mechanism 440);

[0044] Fluorescence detection unit 500 (including fluorescence excitation light source 510 and fluorescence receiving module 520);

[0045] A first reflector 610, a dichroic mirror 620, and a second reflector 630;

[0046] Air supply assembly 700;

[0047] Controller 800;

[0048] Microfluidic chip 900 (including a first microfluidic channel A, a second microfluidic channel B, and a fusion microfluidic channel C). DETAILED DESCRIPTION

[0049] The technical solution of this application is further elaborated in detail below with reference to the accompanying drawings and specific embodiments.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended only to describe specific embodiments and are not intended to limit the implementation of this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0051] In the description of this application, it should be understood that the terms "center," "up," "down," "front," "back," "left," "right," "vertical," "horizontal," "top," "bottom," "inside," "outside," and the like, indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended only to facilitate the description of this application and simplify the description. They do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limiting this application. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0052] In the description of this application, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to direct connections, indirect connections through an intermediate medium, or internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in this application based on the specific circumstances.

[0053] See also Figure 1 and Figure 2 The Y-shaped microfluidic chip 900 used in the droplet fusion system of the present application includes a first microchannel A, a second microchannel B, and a fusion microchannel C. The first microchannel A and the second microchannel B respectively introduce different liquid flows, and after the droplets are generated, they flow in the microchannels; the first microchannel A and the second microchannel B intersect at the fusion microchannel C, and the droplets generated in the first microchannel A and the droplets generated in the second microchannel B are fused in the fusion microchannel C. The fusion process is as follows: Figure 2 As shown in .

[0054] On the microfluidic chip 900, a first laser detection area and a second laser detection area are respectively set on the first microchannel A and the second microchannel B. An image acquisition area is set in the droplet fusion area at the intersection of the first microchannel A, the second microchannel B and the fusion microchannel C. The fusion microchannel C is responsible for droplet fusion and subsequent detection. The fusion microchannel C is provided with a third laser detection area and a fluorescence detection area to ensure that the droplets maintain high quality after fusion and accurately detect their contents. The design of the fusion microchannel C ensures that the droplets can smoothly pass through the subsequent detection areas (the third laser detection area and the fluorescence detection area) after fusion.

[0055] Please refer to Figure 3 and Figure 4In addition to the above-mentioned microfluidic chip 900, the droplet fusion system of one embodiment of the present application also includes a first laser detection unit 100, a second laser detection unit 200, a third laser detection unit 300, an image acquisition unit 400, a fluorescence detection unit 500, an air supply component 700 and a controller 800. The first laser detection unit 100, the second laser detection unit 200, the third laser detection unit 300, the image acquisition unit 400, the fluorescence detection unit 500 and the air supply component 700 are all connected to the controller 800 for signal connection and are centrally and uniformly controlled by the controller 800.

[0056] Among them, the first laser detection unit 100, the second laser detection unit 200, and the third laser detection unit 300 are respectively used to perform laser detection on droplets in the first microfluidic channel A, the second microfluidic channel B, and the fusion microfluidic channel C, respectively, to monitor the physical properties of the droplets and provide high-precision information on droplet size, velocity, and internal particles through laser detection. The image acquisition unit 400 captures images of the fusion channel C in real time, and is used to ensure that the droplet generation and fusion process is in a controllable state by analyzing the droplet size, spacing, velocity, and other indicators in the captured images. It is also used to identify abnormal conditions within the microfluidic channel and achieve real-time detection. The fluorescence detection unit 500 is used to perform fluorescence detection on droplets in the fusion channel C to determine whether the droplets contain the target substance to be captured (such as cells, nucleic acids, etc.). The first microfluidic channel A and the second microfluidic channel B generate droplets through the gas supply component 700. The controller 800 adjusts the gas pressure provided by the gas supply component 700 to the microchannels to control the generation speed and size of the droplets in the first microfluidic channel A and the second microfluidic channel B.

[0057] Laser detection uses the propagation characteristics of light in different media to convert the measured physical quantity into an optical signal. This optical signal is then converted into an electrical signal using a photoelectric converter. After appropriate processing, the corresponding measurement information is obtained. Its specific operating principle is as follows: Due to the different propagation characteristics of light in different media, when a droplet passes through a microfluidic channel, the intensity of the optical signal changes. The frequency and amplitude changes of the optical signal can reflect parameters such as the droplet generation frequency, size, and spacing. An infrared receiver captures these changes and converts them into electrical signals, which are transmitted to the controller 800. The controller 800 receives the digital signal output from the laser detection unit and analyzes the signal using a preset algorithm. By calculating the frequency and amplitude changes of the signal, the droplet generation frequency can be accurately determined.

[0058] For details, please refer to Figure 5The first laser detection unit 100 includes a first infrared laser emitter 110 and a first infrared receiver 120. The first infrared laser emitter 110 is located on one side of the first microchannel A and emits a first detection laser. The first infrared receiver 120 is located on the other side and receives the first feedback light to generate a first laser detection signal. The second laser detection unit 200 includes a second infrared laser emitter 210 and a second infrared receiver 220. The second infrared laser emitter 210 is located on one side of the second microchannel B and emits a second detection laser. The second infrared receiver 220 is located on the other side and receives the second feedback light to generate a second laser detection signal. Similarly, the third laser detection unit 300 includes a third infrared laser emitter 310 and a third infrared receiver 320. The third infrared laser emitter 310 is located on one side of the fusion microchannel C and emits a third detection laser. The third infrared receiver 320 is located on the other side of the fusion microchannel C and receives the third feedback light to generate a third laser detection signal.

[0059] The first infrared laser emitter 110, the second infrared laser emitter 210, and the third infrared laser emitter 310 can emit laser light of a specific wavelength (near-infrared light) and include a special wavelength LED (typically a near-infrared LED), an LED constant current source circuit, a focusing lens, a collimating lens, and a light barrier. The first infrared receiver 120, the second infrared receiver 220, and the third infrared laser receiver 320 include a focusing lens, a filter, a photodetector (such as a silicon photocell, avalanche diode, or a silicon photomultiplier tube), and a signal processing circuit.

[0060] When the droplets pass through the first laser detection area and the second laser detection area of ​​the first microchannel A and the second microchannel B, the controller 800 can use the laser waveform to determine the size, speed and whether there are particles (such as glue balls) inside the droplets after receiving the first laser detection signal and the second laser detection signal from the first infrared receiver 120 and the second infrared receiver 220. This laser detection of a single microchannel can ensure that the droplets are uniform and of high quality before entering the fusion microchannel C, providing a reliable foundation for subsequent fusion operations. The droplet generation frequency of the first microchannel A and the second microchannel B is monitored in real time by the first laser detection unit 100 and the second laser detection unit 200, and the air supply pressure of the air supply component 700 is adjusted by the controller 800. The pressure and droplet flow rate in the first microchannel A and the second microchannel B can be controlled to ensure that the frequency of the two microchannels maintains a 1:1 ratio, creating good prerequisites for subsequent fusion in the fusion microchannel C.

[0061] The third laser detection unit 300 is used to detect the physical properties of droplets in the fused microchannel C after fusion. When droplets pass through the third laser detection zone of the fused microchannel C, the controller 800, upon receiving the third laser detection signal from the third infrared receiver 320, determines whether the droplets in the third laser detection zone have successfully fused. By analyzing the waveform, the controller ensures that the droplet size, velocity, and morphology meet experimental requirements. The third laser detection unit 300 in the fused microchannel C serves as a link between the upper and lower levels, ensuring that droplets entering the subsequent fluorescence detection zone of the fluorescence detection module 500 are intact and reliable.

[0062] The image acquisition unit 400 uses visual recognition technology to capture images of the droplet generation and electrofusion process through the camera device 410, and uses image processing algorithms to analyze the characteristics of the droplets. The specific working principle is as follows: the images of the droplet generation and electrofusion process captured in real time by the camera device 410 are transmitted to the controller 800 (image processing system); after receiving the image information, the controller 800 uses algorithms such as edge detection, image segmentation and feature extraction to identify and analyze the droplet generation frequency, shape, size and electrofusion efficiency; based on preset standards, the controller 800 determines whether there are any abnormalities in the droplet generation and electrofusion process. For example, whether the droplet shape is regular, whether the generation frequency is consistent, whether the fusion efficiency meets the standard, etc. The controller 800 can identify problems that may occur in the droplet generation and electrofusion process through the image information provided by the image acquisition unit 400, and provide timely feedback, such as displaying and issuing reminders through a display device (not shown).

[0063] The camera device 410 is installed below the droplet generation area of ​​the first microchannel A, the second microchannel B, and the fusion area of ​​the fusion microchannel C of the microfluidic chip 900 (the image acquisition area is located at the intersection of the three microchannels). The camera device 410 uses a high-frame rate camera, capable of capturing fast-moving droplets in the image acquisition area. The image acquisition unit 400 can also be equipped with a collimating lens and a fill light to cooperate with the camera device 410. The collimating lens is used to focus light, and the fill light provides sufficient lighting conditions, so that clear images can be obtained even in low-light environments.

[0064] In addition to the camera 410, the image acquisition unit 400 also includes a first adjustment mechanism 420, a second adjustment mechanism 430, and a third adjustment mechanism 440. The camera 410 is mounted on the movable end of the first adjustment mechanism 420, the first adjustment mechanism 420 is mounted on the movable end of the second adjustment mechanism 430, and the second adjustment mechanism 430 is mounted on the movable end of the third adjustment mechanism 440. The first adjustment mechanism 420, the second adjustment mechanism 430, and the third adjustment mechanism 440 can each move their movable ends in three different directions, thereby allowing the position of the camera 410 to be adjusted in three directions. This allows the camera 410 to have a wider range of images within a certain range and adjust the shooting area and shooting distance of the camera 410. In the illustrated embodiment, the movable ends of the first adjustment mechanism 420, the second adjustment mechanism 430, and the third adjustment mechanism 440 move in perpendicular directions, i.e., in the Z direction (up and down), the Y direction (forward and backward), and the X direction (left and right), respectively, to achieve three-dimensional adjustment.

[0065] The camera device 410 in the image acquisition unit 400 can use a large-area CCD module to capture the images of the droplets in the three pipes of the first microchannel A, the second microchannel B, and the fusion microchannel C in real time. The controller 800 assists other detection modules by analyzing indicators such as the size, spacing, and speed of the droplets to ensure that the droplet generation and fusion process is in a controllable state. The image acquisition unit 400 can also identify abnormal conditions in the microchannel, such as bubbles, foreign matter, droplet jams, or pipe blockages. Since these problems are difficult to detect through laser detection modules or fluorescence detection modules, the image analysis of the image acquisition unit 400 becomes an indispensable monitoring means, providing more comprehensive protection for experimental operations. Therefore, the image analysis of the image acquisition unit 400 can further verify the morphology and flow state of the droplets, and at the same time discover abnormal problems that other detection methods cannot identify.

[0066] The fluorescence detection unit 500 is used to determine whether the fused droplets contain the target substance to be captured, and includes a fluorescence excitation light source 510 and a fluorescence receiving module 520. The fluorescence excitation light source 510 is arranged on one side of the fusion microchannel C and emits fluorescence excitation light, and the fluorescence receiving module 520 is installed on the other side and receives the excited fluorescence to generate a fluorescence detection signal. During the experiment, the target cells or nucleic acids are pre-labeled with fluorescent dyes. When these markers enter the droplets and flow through the fluorescence detection area of ​​the fusion microchannel C, the fluorescence excitation light source 510 emits fluorescence excitation light to illuminate the droplets in the fluorescence detection area, and judges whether the droplets contain the target substance based on the fluorescence received by the fluorescence receiving module 520. The intensity of the fluorescence received by the fluorescence receiving module 520 can directly reflect the concentration or quantity of the target substance in the droplets.

[0067] Since the size of the microfluidic chip 900 is small and the intervals between the first microchannel A, the second microchannel B, and the fusion microchannel C are small, how to arrange the above-mentioned first laser detection unit 100, the second laser detection unit 200, the third laser detection unit 300, the image acquisition unit 400, and the fluorescence detection unit 500 in the limited space around the microfluidic chip 900 without interfering with each other is also a key technical point to be solved.

[0068] Please refer to further Figure 5 、 Figure 6 and Figure 7 The microfluidic chip 900 is horizontally arranged on a plane where the X and Y directions are located, and the first laser detection unit 100, the second laser detection unit 200, the third laser detection unit 300, the image acquisition unit 400 and the fluorescence detection unit 500 are arranged around the microfluidic chip 900.

[0069] Specifically, the first infrared laser emitter 110, the second infrared laser emitter 310, the third infrared laser emitter 310, and the fluorescence excitation light source 510 are all disposed above the microfluidic chip 900 (higher than the microfluidic chip 900 in the Z direction), so that the emitted first detection laser, second detection laser, third detection laser, and fluorescence excitation light are all irradiated from above to each microchannel of the microfluidic chip 900. Correspondingly, the first infrared receiver 120, the second infrared receiver 220, the third infrared receiver 320, and the fluorescence receiving module 520 are all disposed below the microfluidic chip 900 (lower than the microfluidic chip 900 in the Z direction).

[0070] More specifically, the first infrared laser emitter 110 and the second infrared laser emitter 310 are positioned opposite each other in the front-to-back direction, emitting a first detection laser and a second detection laser, respectively, along the Y direction. To ensure that the emitted detection lasers are directed toward the microfluidic chip 900 along the Z direction, a first reflector 610 is positioned above the first laser detection zone of the first microchannel A and the second laser detection zone of the second microchannel B. The first reflector 610 reflects the first and second detection lasers from both sides in the Y direction into beams directed downward along the Z direction. The first reflector 610 has a first reflective surface that reflects the first detection laser light emitted by the first infrared laser emitter 110 in the Y direction so that it is directed toward the first microchannel A in the Z direction. The first reflector 610 has a second reflective surface that reflects the second detection laser light emitted by the second infrared laser emitter 110 in the Y direction so that it is directed toward the second microchannel B in the Z direction. In the illustrated embodiment, the first reflector 610 can be a prism, formed by combining a prism having a first reflective surface and a prism having a second reflective surface.

[0071] More specifically, the third infrared laser emitter 310 is disposed directly above the third laser detection area of ​​the fusion microchannel C.

[0072] As previously described, the camera device 410 is positioned below the image acquisition area at the intersection of the first microchannel A, the second microchannel B, and the fusion microchannel C, and is movable in the X, Y, and Z directions. Therefore, the first infrared receiver 120, the second infrared receiver 220, and the third infrared receiver 320 located below the microfluidic chip 900 must be positioned away from the image acquisition area. To this end, a dichroic mirror 620 is positioned below the image acquisition area of ​​the microfluidic chip 900 and between the camera device 410. The dichroic mirror 620 is tilted at a 45° angle to the Z direction, reflecting infrared light and transmitting white light. Specifically, the dichroic mirror 620 reflects the first, second, and third feedback beams of the first, second, and third detection laser beams, which pass through the microchannels in the downward Z direction, forward in the Y direction, while transmitting the white light in the Z direction, enabling the camera device 410 to capture images of the image acquisition area.

[0073] The compact space prevents the first infrared receiver 120, the second infrared receiver 220, and the third infrared receiver 320 from being simultaneously arranged in the Y direction. Therefore, the third infrared receiver 320 is positioned in the Y direction directly opposite the reflective surface of the dichroic mirror 620, while the first infrared receiver 120 and the second infrared receiver 220 are positioned on either side of the X direction. A second reflector 630 is positioned in the Y direction of the reflective surface of the dichroic mirror 620. This reflector 630 reflects the first and second feedback lights from the dichroic mirror 620 in the Y direction, directing them to propagate in both directions along the X direction. The second reflector 630 has a third reflective surface that reflects the first detection laser light emitted by the first infrared laser emitter 110, which passes through the first reflector 610, the first microchannel A, and the dichroic mirror 620, along the X direction toward the first infrared receiver 120 for reception by the first infrared receiver 120. The second reflector 630 has a fourth reflective surface that reflects the second detection laser light emitted by the second infrared laser emitter 210, which passes through the first reflector 610, the first microchannel B, and the dichroic mirror 620, along the X direction toward the second infrared receiver 220 for reception by the second infrared receiver 220. In the illustrated embodiment, the second reflector 630 may be a prism, formed by combining a prism having a third reflective surface and a prism having a fourth reflective surface.

[0074] More specifically, the fluorescence excitation light source 510 is disposed directly above the fluorescence detection area of ​​the fusion microchannel C, the fluorescence receiving module 520 is disposed directly below the fluorescence detection area, and the fluorescence receiving module 520 is aligned with the fluorescence excitation light source 510 in the Z direction.

[0075] In the droplet fusion system of the present application, laser detection units (a first laser detection unit 100, a second laser detection unit 200, and a third laser detection unit 300) and a fluorescence detection unit 500 are provided. At the same time, the image analysis technology of the image acquisition unit 400 is used to realize global real-time monitoring. Through the coordinated work of different detection means, the droplet operation during the experiment is ensured to be accurate and stable, and the experimental parameters can be automatically adjusted to optimize the experimental conditions. The droplet fusion system of the present application can monitor the droplet generation frequency: the first laser detection unit 100 and the second laser detection unit 200 detect the droplet generation frequency of the first microchannel A and the second microchannel B in real time. When the droplets pass through the first laser detection area and the second laser detection area, the changes in the light signal are captured by the infrared receiver and transmitted to the processor 800. The processor 800 analyzes the frequency changes of the light signal and calculates the droplet generation frequency and droplet size of the first microchannel A and the second microchannel B. Based on the analysis of the droplet signals, the controller 800 adjusts the flow rate and pressure of the first microchannel A and the second microchannel B in real time through the gas supply assembly 700 to ensure that the stability and frequency of droplet generation in the first microchannel A and the second microchannel B maintain a 1:1 ratio. This real-time, high-precision detection and adjustment method ensures that the droplets reach their optimal state before electrofusion.

[0076] The droplet fusion system of the present application can monitor the fusion process: the image acquisition unit 400 takes the droplet image of the electrofusion area (image acquisition area) in real time, and the controller 800 detects the shape, size and fusion status of the droplet through the image analysis algorithm, identifies the morphology of the droplet after fusion in real time, and analyzes the efficiency and success rate of electrofusion. Specifically, the image acquisition unit 400 takes the droplet image of the first microchannel A and the second microchannel B in real time, and the controller 800 extracts the outline and generation frequency of the droplet through edge detection and image segmentation. When the droplet generation frequency and droplet size of the first microchannel A and the second microchannel B do not meet the preset conditions, the controller 800 will automatically adjust the pressure of the corresponding microchannel through the air supply component 700 according to the frequency and size until the preset value is met.

[0077] Furthermore, the droplet generation frequency data provided by the first and second laser detection units 100 and 200 is integrated with the fused image data provided by the image acquisition unit 400 and then fed into the controller 800. This data is then comprehensively analyzed to determine whether the droplet generation frequencies of the first and second microchannels A and B are consistent. If not, the gas supply assembly 700 automatically adjusts the flow rate and pressure of the first and second microchannels A and B to ensure a 1:1 frequency ratio between the two microchannels, further improving the accuracy of monitoring and adjustment.

[0078] When the controller 800 detects inconsistent droplet generation frequency or low fusion efficiency, it immediately issues a warning and displays relevant images and parameters on the display. Maintenance personnel, following the prompts on the display, promptly adjust relevant droplet generation and fusion parameters, such as flow rate, pressure, and electric field strength. The display shows the adjusted parameters and the results, ensuring optimal droplet generation frequency and fusion efficiency.

[0079] In the droplet fusion system of this application, a laser detection unit, an image acquisition unit, and a fluorescence detection unit are used to achieve quality control during the droplet generation and fusion process. This not only monitors and adjusts the droplet generation frequency and fusion efficiency, but also takes advantage of the following complementary advantages of various technologies:

[0080] (1) High sensitivity of laser detection: The laser detection unit is very sensitive to changes in refractive index, especially when the droplet is wrapped with a gel ball containing a special DNA tag. Since the gel ball material (such as hydrogel) is nearly transparent, the imaging method may not be able to effectively identify the DNA-tagged gel ball wrapped in the oil-in-water structure, which may lead to misjudgment. Laser detection can accurately detect the refractive index changes inside the droplet, thereby accurately identifying the encapsulated DNA-tagged gel ball.

[0081] (2) High response speed of laser detection: The fastest response speed of laser detection technology can reach picosecond level (10 -12 seconds), and the response speed of visual recognition technology using general industrial cameras (small-sized cameras) as detection devices is about milliseconds (10 -3 Seconds), laser detection technology makes up for the problem of insufficient response speed of high-speed droplets under low cost and small space.

[0082] (3) Intuitiveness of image acquisition: The image acquisition unit can intuitively detect the shape, size and fusion of droplets through image analysis, which is very important for detecting the physical state of droplets and the morphological changes after fusion.

[0083] (4) Data fusion and comprehensive analysis: The droplet generation frequency and internal structure data provided by the laser detection unit are combined with the droplet morphology and fusion efficiency data provided by the image acquisition unit, and the controller performs a comprehensive analysis. The monitoring data of the laser detection unit and the monitoring data of the image acquisition unit can ensure the accuracy of the detection through cross-validation data. The physical information such as the size and speed of the droplets can be calibrated with each other through joint detection to ensure the accuracy of the droplet physical property data, so that abnormal conditions in the droplet generation and fusion process can be judged more accurately and timely adjustment suggestions can be provided. In addition, the detection data of the fluorescence detection unit can be further combined with physical data to confirm whether the droplets contain the target substance, thereby reducing false positives.

[0084] In summary, the present application provides a droplet fusion system, which combines a laser detection unit, an image acquisition unit, and a fluorescence detection unit, and proposes a multiple, multi-level quality control scheme to achieve real-time and accurate monitoring of the droplet generation and fusion process. It can also perform real-time and automated quality control by adjusting parameters, significantly improving the reliability and efficiency of single-cell sequencing technology.

[0085] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0086] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A droplet fusion system, characterized in that: include: A microfluidic chip comprising a first microchannel, a second microchannel, and a fused microchannel, wherein the first microchannel and the second microchannel merge into the fused microchannel, a first laser detection area and a second laser detection area are formed on the first microchannel and the second microchannel, respectively, and an image acquisition area is formed at the intersection of the first microchannel, the second microchannel, and the fused microchannel; A gas supply component, used for injecting gas into the first microchannel and the second microchannel; a first laser detection unit, configured to emit a first detection laser, receive a first feedback light formed after passing through the first laser detection area, and generate a first laser detection signal; a second laser detection unit, configured to emit a second detection laser, receive a second feedback light formed after passing through the second laser detection area, and generate a second laser detection signal; An image acquisition unit, configured to capture an image of the image acquisition area; a controller that receives detection signals from the first laser detection unit and the second laser detection unit and image information from the image acquisition unit, and controls the gas supply assembly to adjust the speed and pressure of the liquid flow in the first microchannel and the second microchannel based on the first laser detection signal, the second laser detection signal, and the image information; The first laser detection unit includes a first infrared laser emitter and a first infrared receiver; the first infrared laser emitter is located on one side of the first microchannel and emits the first detection laser, and the first infrared receiver is located on the other side to receive the first feedback light and generate the first laser detection signal; The second laser detection unit includes a second infrared laser emitter and a second infrared receiver; the second infrared laser emitter is located on one side of the second microchannel and emits the second detection laser, and the second infrared receiver is located on the other side to receive the second feedback light and generate the second laser detection signal; After receiving the first laser detection signal and the second laser detection signal, the controller uses the laser waveform to determine the size and speed of the droplets in the first microchannel and the second microchannel, as well as whether there are particles inside; the controller uses the image information to identify and analyze the generation frequency, shape, size and electrofusion efficiency of the droplets.

2. The droplet fusion system according to claim 1, characterized in that: Also includes: The third laser detection unit is used to emit a third laser signal, receive third feedback light formed after passing through the third laser detection area on the fusion microchannel, and generate a third laser detection signal.

3. The droplet fusion system according to claim 2, characterized in that: Also includes: The fluorescence detection unit is used to emit fluorescence excitation light, receive the fluorescence excited after passing through the fluorescence detection area on the fusion microchannel, and generate a fluorescence detection signal.

4. The droplet fusion system according to claim 3, characterized in that: The third laser detection unit includes a third infrared laser emitter and a third infrared receiver; the image acquisition unit includes a camera device, and the camera device is below the image acquisition area; the fluorescence detection unit includes a fluorescence excitation light source and a fluorescence receiving module; Among them, the first infrared laser emitter, the second infrared laser emitter, the third infrared laser emitter and the fluorescence excitation light source are all arranged above the microfluidic chip, and the first infrared receiver, the second infrared receiver, the third infrared receiver and the fluorescence receiving module are all arranged below the microfluidic chip.

5. The droplet fusion system according to claim 4, characterized in that: The image acquisition unit also includes a first adjustment mechanism, a second adjustment mechanism and a third adjustment mechanism. The camera device is installed at the movable end of the first adjustment mechanism, the first adjustment mechanism is installed at the movable end of the second adjustment mechanism, and the second adjustment mechanism is installed at the movable end of the third adjustment mechanism. The moving directions of the movable end of the first adjustment mechanism, the movable end of the second adjustment mechanism and the movable end of the third adjustment mechanism are perpendicular to each other.

6. The droplet fusion system according to claim 4, characterized in that: A first reflective element is arranged above the first laser detection area and the second laser detection area. The first infrared laser emitter and the second infrared laser emitter are relatively arranged on both sides of the first reflective element in the front-to-back direction. The first reflective element reflects the first detection laser and the second detection laser downward.

7. The droplet fusion system according to claim 6, characterized in that: The third infrared laser emitter is arranged directly above the third laser detection area.

8. The droplet fusion system according to claim 7, characterized in that: A dichroic mirror is provided below the image acquisition area and between the camera device. The dichroic mirror transmits white light and reflects the first feedback light, the second feedback light, and the third feedback light directed downwards forward.

9. The droplet fusion system according to claim 8, characterized in that: The third infrared receiver is arranged in the front-to-back direction and faces the dichroic mirror. A second reflector is arranged in front of the dichroic mirror. The first infrared receiver and the second infrared receiver are respectively arranged on the left and right sides of the second reflector.

10. The droplet fusion system according to claim 4, characterized in that: The fluorescence excitation light source is arranged directly above the fluorescence detection area, and the fluorescence receiving module is aligned with the fluorescence excitation light source in the vertical direction.

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