Droplet fusion system

Abstract

The invention provides a liquid drop fusion system. The liquid drop fusion system comprises a micro-fluidic chip; an air supply assembly; the first laser detection unit is used for emitting first detection laser, receiving first feedback light formed after the first detection laser passes through a first laser detection area and generating a first laser detection signal; the second laser detection unit is used for emitting second detection laser, receiving second feedback light formed after the second detection laser passes through a second laser detection area and generating a second laser detection signal; the image acquisition unit is used for shooting an image of the image acquisition area; and the controller is used for receiving detection signals of the first laser detection unit and the second laser detection unit and image information of the image acquisition unit, and controlling the gas supply assembly to adjust the speed and pressure of liquid flows in the first micro-channel and the second micro-channel. According to the droplet fusion system, the laser detection unit and the image acquisition unit are adopted to monitor the droplet generation and fusion process, the controller adjusts parameters for real-time quality control, and the reliability and efficiency of the single cell sequencing technology are remarkably improved.

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 bioanalysis tool, droplet microfluidics has been widely used in single-cell analysis, digital PCR, immunoassay and other fields. By encapsulating single cells or reaction components in tiny oil droplets, droplet technology provides unprecedented possibilities for high-throughput parallel processing. However, with the deepening of life science research and the increase in application needs, simple droplet encapsulation technology has gradually shown its limitations and is difficult 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 inside the droplets to achieve more complex and sophisticated experimental designs.

[0004] For example, in large-scale biological analysis and drug screening, not only high throughput is required, but also content exchange and mixing between multiple droplets is required to simulate complex biological reaction environments. This demand places higher demands on droplet technology, which needs to be able to process and fuse a large number of droplets with different components in a short time and ensure the consistency and reliability of each operation.

[0005] In view of the growing demand for the above applications, droplet fusion technology has emerged. Droplet fusion technology can fuse different droplet contents together to perform more complex chemical reactions, biological analysis and material synthesis. This technology can not only greatly improve the flexibility and accuracy of the experiment, but also integrate a variety of different experiments on one platform, greatly expanding the application scope of droplet microfluidics technology.

[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 in droplets. On the other hand, by performing multiple reaction steps in a single droplet, droplet fusion technology can significantly reduce experimental operation time and reagent consumption, which not only improves experimental efficiency but also reduces costs, which is particularly 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 applications:

[0008] (1) Unstable fusion success rate: Instability in the droplet fusion process is still a major problem. If the fusion efficiency is low or the fusion is 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 depends 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 be discovered and corrected 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 purpose, the technical solution of the embodiment of the present application is implemented as follows:

[0012] The embodiment of the present application 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, used for emitting a second detection laser, receiving a second feedback light formed after passing through the second laser detection area, and generating a second laser detection signal;

[0017] An image acquisition unit, used for capturing an image of the image acquisition area;

[0018] A controller receives 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 controls the gas supply component to adjust the speed and pressure of the liquid flow in the first microchannel and the second microchannel according to the first laser detection signal, the second laser detection signal and the image information.

[0019] In one embodiment, it also includes:

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

[0021] In one embodiment, it also 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 transmitter and a first infrared receiver; the second laser detection unit includes a second infrared laser transmitter and a second infrared receiver; the third laser detection unit includes a third infrared laser transmitter 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 fluorescent 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 fluorescent receiving module are all arranged below the microfluidic chip.

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

[0026] In one embodiment, a first reflective element is disposed 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 disposed 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 of the embodiments, the third infrared laser emitter is disposed directly above the third laser detection area.

[0028] In one of the embodiments, a dichroic mirror is arranged 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 both sides of the second reflective element in the left and right directions.

[0030] In one of the embodiments, 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 the up-down 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 the change of the refractive index and has the advantage of fast response speed, so that it can accurately detect the refractive index change 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 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, which significantly improves the reliability and efficiency of the 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 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 5A 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 schematic diagram of 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 labeled as follows:

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

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

[0042] A third laser detection unit 300 (including a third infrared laser transmitter 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 fused microfluidic channel C). DETAILED DESCRIPTION

[0049] The technical solution of the present application is further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments of the specification.

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

[0051] In the description of the present application, it should be understood that the terms "center", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating positions or positional relationships, are based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific position, be constructed and operated in a specific position, and therefore cannot be understood as a limitation on the present application. In the description of the present application, unless otherwise specified, "multiple" means two or more.

[0052] In the description of this application, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection, or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to 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 microfluidic channel A, a second microfluidic channel B and a fusion microfluidic channel C. The first microfluidic channel A and the second microfluidic channel B respectively introduce different liquid flows, and the droplets are generated and flow in the microchannels; the first microfluidic channel A and the second microfluidic channel B intersect at the fusion microchannel C, and the droplets generated in the first microfluidic channel A and the droplets generated in the second microfluidic channel 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, the first microchannel A and the second microchannel B are respectively provided with a first laser detection area and a second laser detection area. An image acquisition area is provided 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 remain of 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 (third laser detection area and 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 an 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 by signals 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 used to perform laser detection on the droplets in the first microchannel A, the second microchannel B, and the fusion microchannel C respectively, for monitoring the physical properties of the droplets, and providing high-precision droplet size, speed and internal particle information through laser detection; the image acquisition unit 400 takes pictures and collects images of the fusion channel C in real time, and is used to ensure that the generation and fusion process of the droplets is in a controllable state by analyzing the size, spacing, speed and other indicators of the droplets in the collected images, and is also used to identify abnormal conditions in the microchannel to achieve real-time detection; the fluorescence detection unit 500 is used to perform fluorescence detection on the droplets in the fusion channel C, and is used to determine whether the droplets contain the target substance to be captured (such as cells, nucleic acids, etc.). The first microchannel A and the second microchannel B generate droplets by supplying gas to the inside through the gas supply component 700, and the generation speed and size of the droplets in the first microchannel A and the second microchannel B can be controlled by adjusting the gas pressure provided by the gas supply component 700 to the microchannels through the controller 800.

[0057] Laser detection uses the propagation characteristics of light in different media to convert the measured physical quantity into an optical signal, and then uses a photoelectric converter to convert the optical signal into an electrical signal. After corresponding processing, the corresponding measurement information is obtained. Its specific working principle is as follows: Due to the different propagation characteristics of light between different media, when a droplet passes through a microchannel, the intensity of the optical signal will change. The frequency and amplitude changes of the optical signal can reflect the parameters such as the generation frequency, size and spacing of the droplets; the infrared receiver captures these changes and converts them into electrical signals and transmits them to the controller 800; the controller 800 receives the digital signal output from the laser detection unit, analyzes the signal using a preset algorithm, and calculates the frequency and amplitude changes of the signal to accurately obtain the generation frequency of the droplets.

[0058] For details, please refer to Figure 5, the 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 transmitter 110, the second infrared laser transmitter 210 and the third infrared laser transmitter 310 can emit laser light of a specific wavelength (near infrared light), and include a special wavelength LED (usually a near infrared LED), an LED constant current source circuit, a focusing lens, a collimating lens and a light barrier, etc. 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, an avalanche diode, a silicon photomultiplier tube, etc.), a signal processing circuit, etc.

[0060] When the droplet passes 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 judge the size, speed and whether there are particles (such as glue balls) inside the droplet by the laser waveform after receiving the first laser detection signal and the second laser detection signal of the first infrared receiver 120 and the second infrared receiver 220. This laser detection of a single microchannel can ensure that the droplet is uniform and of high quality before entering the fusion microchannel C, providing a reliable basis 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 the droplets in the fused microchannel C after fusion. When the droplets pass through the third laser detection area of ​​the fused microchannel C, the controller 800 determines whether the droplets in the third laser detection area are successfully fused after receiving the third laser detection signal from the third infrared receiver 320, and ensures that the droplet size, speed and shape meet the experimental requirements by analyzing the waveform. The third laser detection unit 300 on the fused microchannel C plays a connecting role, ensuring that the droplets entering the fluorescence detection area of ​​the subsequent fluorescence detection module 500 are complete 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 the image processing algorithm to analyze the characteristics of the droplets. The specific working principle is as follows: the images of the droplet generation and electrofusion process taken 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 identifies and analyzes the generation frequency, shape, size and electrofusion efficiency of the droplets through algorithms such as edge detection, image segmentation and feature extraction; according to the preset standards, the controller 800 determines whether there are 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 is up to standard, etc. The controller 800 can identify the 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 and can capture 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 in conjunction with the camera device 410. The collimating lens is used to focus light, and the fill light provides sufficient lighting conditions, so that a clear image can be obtained even in a low-light environment.

[0064] In addition to the camera device 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 device 410 is installed at the movable end of the first adjustment mechanism 420, the first adjustment mechanism 420 is installed at the movable end of the second adjustment mechanism 430, and the second adjustment mechanism 430 is installed at 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 move the movable ends in three different directions respectively, so that the position of the camera device 410 can be adjusted in three directions, so that the shooting area and shooting distance of the camera device 410 can be adjusted, and a larger shooting range can be achieved within a certain range. In the illustrated embodiment, the moving directions of the movable ends of the first adjustment mechanism 420, the second adjustment mechanism 430 and the third adjustment mechanism 440 are perpendicular to each other, that is, they are respectively arranged in the Z direction (up and down direction), the Y direction (front and back direction), and the X direction (left and right direction) 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 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 the size, spacing, speed and other indicators of the droplets to ensure that the generation and fusion process of the droplets are 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. Among them, the fluorescence excitation light source 510 is arranged on one side of the fusion microfluidic channel 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 microfluidic channel 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 fused 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 direction and the Y direction 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 fluorescent excitation light source 510 are all arranged 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 fluorescent 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 fluorescent receiving module 520 are all arranged 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 arranged opposite to each other in the front-back direction, and emit the first detection laser and the second detection laser respectively in the Y direction. In order to realize that the emitted detection laser is irradiated onto the microfluidic chip 900 in the Z direction, a first reflector 610 is arranged above the first laser detection area of ​​the first microchannel A and the second laser detection area of ​​the second microchannel B. The first reflector 610 reflects the first detection laser and the second detection laser from both sides in the Y direction into a beam downward in the Z direction. The first reflector 610 has a first reflection surface, which reflects the first detection laser emitted by the first infrared laser emitter 110 in the Y direction to irradiate the first microchannel A in the Z direction; the first reflector 610 has a second reflection surface, which reflects the second detection laser emitted by the second infrared laser emitter 110 in the Y direction to irradiate the second microchannel B in the Z direction. In the illustrated embodiment, the first reflector 610 can be a prism, which is composed of a prism having a first reflection surface and a prism having a second reflection 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 mentioned above, the camera device 410 is arranged below the image acquisition area at the intersection of the first microchannel A, the second microchannel B and the fusion microchannel C, and can move in the X direction, the Y direction and the Z direction. Therefore, the first infrared receiver 120, the second infrared receiver 220 and the third infrared receiver 320 located below the microfluidic chip 900 need to be arranged away from the position below the image acquisition area. Based on this, a dichroic mirror 620 is arranged between the image acquisition area below the microfluidic chip 900 and the camera device 410. The dichroic mirror 620 is arranged at a 45° inclination with the Z direction, which can reflect infrared light and transmit white light. That is, the dichroic mirror 620 reflects the first feedback light, the second feedback light and the third feedback light of the first detection laser, the second detection laser and the third detection laser downward along the Z direction after passing through the microchannel to the forward direction along the Y direction, and transmits the white light along the Z direction, so that the camera device 410 can capture images of the image acquisition area.

[0073] The compact space makes it impossible to arrange the first infrared receiver 120, the second infrared receiver 220 and the third infrared receiver 320 in the Y direction at the same time. Therefore, the third infrared receiver 320 is arranged in the Y direction directly facing the reflection surface of the dichroic mirror 620, and the first infrared receiver 120 and the second infrared receiver 220 are arranged on both sides of the X direction. In the Y direction of the reflection surface of the dichroic mirror 620, a second reflector 630 is arranged. The second reflector 630 reflects the first feedback light and the second feedback light from the dichroic mirror 620 in the Y direction to propagate to both sides in the X direction. The second reflector 630 has a third reflective surface, and reflects the light of the first detection laser emitted by the first infrared laser emitter 110, which is reflected by the first reflector 610, the first microchannel A, and the dichroic mirror 620, to the first infrared receiver 120 along the X direction, so as to be received by the first infrared receiver 120; the second reflector 630 has a fourth reflective surface, and reflects the light of the second detection laser emitted by the second infrared laser emitter 210, which is reflected by the first reflector 610, the first microchannel B, and the dichroic mirror 620, to the second infrared receiver 220 along the X direction, so as to be received by the second infrared receiver 220. In the illustrated embodiment, the second reflector 630 may be a prism, which is composed of 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 (first laser detection unit 100, second laser detection unit 200 and third laser detection unit 300) and fluorescence detection unit 500 are provided, and the image analysis technology of 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 optical signal are captured by the infrared receiver and transmitted to the processor 800. The processor 800 analyzes the frequency changes of the optical signal and calculates the droplet generation frequency and droplet size of the first microchannel A and the second microchannel B. According to the analysis results of the droplet signal, 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 component 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 can ensure that the droplets reach the 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 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 contour 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] In addition, after the droplet generation frequency data provided by the first laser detection unit 100 and the second laser detection unit 200 are integrated with the fusion image data provided by the image acquisition unit 400, the controller 800 comprehensively analyzes the droplet generation frequency and fusion efficiency to confirm whether the droplet generation frequency of the first microchannel A and the second microchannel B is consistent. If not, the flow rate and pressure of the first microchannel A and the second microchannel B are automatically adjusted through the gas supply component 700 to ensure that the frequency of the two microchannels remains 1:1, thereby further improving the accuracy of monitoring and adjustment.

[0078] When the controller 800 analyzes that the droplet generation frequency is inconsistent or the fusion efficiency is low, it will immediately issue a warning prompt and display relevant images and parameters on the display device. Maintenance personnel will adjust the relevant parameters of droplet generation and fusion, such as flow rate, pressure and electric field strength, in a timely manner according to the prompts of the display device. The display device displays the adjusted relevant parameters and shows the adjustment effect to ensure that the droplet generation frequency and fusion efficiency are in the optimal state.

[0079] In the droplet fusion system of the present 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, which is not only to monitor and adjust the droplet generation frequency and fusion efficiency, but also to have the following complementary advantages of multiple 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 colloid containing a special DNA tag. Since the material of the colloid (such as hydrogel) is nearly transparent, the image method may not be able to effectively identify the DNA-tagged colloid 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 wrapped DNA-tagged colloid.

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

[0082] (3) Intuitive 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 by cross-validating the 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 alarms.

[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, and can 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 article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0086] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope 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 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, used for emitting a second detection laser, receiving a second feedback light formed after passing through the second laser detection area, and generating a second laser detection signal; An image acquisition unit, used for capturing an image of the image acquisition area; A controller receives 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 controls the gas supply component to adjust the speed and pressure of the liquid flow in the first microchannel and the second microchannel according to the first laser detection signal, the second laser detection signal and the image information.

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 a third feedback light formed after passing through a 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 first laser detection unit includes a first infrared laser transmitter and a first infrared receiver; the second laser detection unit includes a second infrared laser transmitter and a second infrared receiver; the third laser detection unit includes a third infrared laser transmitter 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 fluorescent 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 fluorescent 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 ends of the first adjustment mechanism, the second adjustment mechanism and 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 arranged 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 directly 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 both sides of the second reflector in the left and right directions.

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 up-down direction.

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