Droplet fusion device

Abstract

The utility model provides liquid drop fusion equipment. The liquid drop fusion equipment comprises a micro-fluidic chip; the first laser detection unit is used for emitting first detection laser, receiving the first feedback light and generating a first laser detection signal; the second laser detection unit is used for emitting second detection laser, receiving second feedback light and generating a second laser detection signal; the image acquisition unit is used for shooting an image of the image acquisition area; the fluorescence detection unit is used for emitting fluorescence excitation light, receiving excited fluorescence and generating a fluorescence detection signal; 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 adjusting the speed and the pressure of liquid flows in the first micro-channel and the second micro-channel according to the detection signals and the image information. According to the droplet fusion equipment, 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 technical field of single-cell sequencing, and in particular to a droplet fusion device. BACKGROUND

[0002] As an important tool for biological analysis, 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, as life science research deepens and application demands increase, the simple droplet encapsulation technology has gradually shown its limitations, making it difficult to meet the increasingly complex experimental requirements.

[0003] For example, the development of modern biotechnology not only requires encapsulating samples in droplets for simple independent reactions, but also requires 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 delicate experimental designs.

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

[0005] In view of the growing application requirements, 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 not only greatly improves the flexibility and accuracy of experiments, but also enables the integration of multiple different experiments on one platform, greatly expanding the application range of droplet microfluidics.

[0006] On the one hand, droplet fusion technology allows researchers to fuse samples and reagents within different droplets, enabling multi-step reactions or multi-sample interactions, which greatly improves the complexity and flexibility of experiments, enabling reactions that would otherwise be performed in steps to be completed continuously within droplets. On the other hand, by performing multiple reaction steps within 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 analysis results, which is particularly important in gene editing, single-molecule detection, and complex sample analysis that require fine control.

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

[0008] (1) Fusion success rate is unstable: the instability in the droplet fusion process is still a major problem. If the fusion efficiency is low or incomplete, it will lead to sample loss, data bias, and affect the reliability of experimental results.

[0009] (2) Lack of real-time monitoring means: Currently, the success of droplet fusion often depends on post-analysis, and there is a lack of effective real-time monitoring means 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

[0010] To solve the existing technical problems, the present application provides a droplet fusion device to realize real-time monitoring of droplet generation frequency and electric fusion process.

[0011] To achieve the above purpose, the technical scheme of the embodiments of the present application is as follows:

[0012] The present application provides a droplet fusion device, comprising:

[0013] A microfluidic chip comprising a first microfluidic channel, a second microfluidic channel and a fusion microfluidic channel, the first microfluidic channel and the second microfluidic channel converge at the fusion microfluidic channel, the first microfluidic channel and the second microfluidic channel form a first laser detection area and a second laser detection area respectively, and the intersection of the first microfluidic channel, the second microfluidic channel and the fusion microfluidic channel forms an image acquisition area;

[0014] A first laser detection unit for emitting first detection laser, receiving first feedback light formed after passing through the first laser detection area and generating first laser detection signal;

[0015] A second laser detection unit for emitting second detection laser, receiving second feedback light formed after passing through the second laser detection area and generating second laser detection signal;

[0016] An image acquisition unit for shooting images of the image acquisition area;

[0017] A fluorescence detection unit for emitting fluorescence excitation light, receiving fluorescence excited after passing through a fluorescence detection area on the fusion microfluidic channel and generating fluorescence detection signal;

[0018] A controller 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 adjusting the speed and pressure of the liquid flow in the first microfluidic channel and the second microfluidic channel accordingly.

[0019] In one of the embodiments, the first laser detection unit comprises a first infrared laser emitter and a first infrared receiver; the second laser detection unit comprises a second infrared laser emitter and a second infrared receiver; the image acquisition unit comprises a camera, which is below the image acquisition area; the fluorescence detection unit comprises a fluorescence excitation light source and a fluorescence receiving module;

[0020] In one of the embodiments, the first infrared laser emitter, the second infrared laser emitter and the fluorescence excitation light source are arranged above the microfluidic chip, and the first infrared receiver, the second infrared receiver and the fluorescence receiving module are arranged below the microfluidic chip.

[0021] In one of the embodiments, the image acquisition unit further comprises a first adjusting mechanism, a second adjusting mechanism and a third adjusting mechanism, the camera is mounted on the movable end of the first adjusting mechanism, the first adjusting mechanism is mounted on the movable end of the second adjusting mechanism, the second adjusting mechanism is mounted on the movable end of the third adjusting mechanism, and the moving directions of the movable ends of the first adjusting mechanism, the second adjusting mechanism and the third adjusting mechanism are perpendicular to each other.

[0022] In one of the embodiments, the device further comprises:

[0023] A third laser detection unit for emitting a third laser signal, receiving a third feedback light formed after passing through a third laser detection area on the fusion microfluidic channel, and generating a third laser detection signal.

[0024] In one of the embodiments, the third laser detection unit comprises a third infrared laser emitter and a third infrared receiver; the third infrared laser emitter is arranged above the microfluidic chip, and the third infrared receiver is arranged below the microfluidic chip.

[0025] In one of the embodiments, a first reflector 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 arranged on the two sides of the first reflector in the front-rear direction, and the first reflector reflects the first detection laser and the second detection laser downward.

[0026] In one of the embodiments, the third infrared laser emitter is arranged directly above the third laser detection area.

[0027] In one of the embodiments, a dichroic mirror is arranged between the image acquisition area and the camera, the dichroic mirror transmits white light and reflects the first feedback light, the second feedback light and the third feedback light downward.

[0028] In one embodiment, the third infrared receiver is positioned in the front-to-back direction and directly opposite the dichroic mirror, a second reflector is positioned in front of the dichroic mirror, and the first infrared receiver and the second infrared receiver are respectively positioned on the left and right sides of the second reflector.

[0029] In one embodiment, the fluorescence excitation light source is positioned directly above the fluorescence detection area, and the fluorescence receiving module is aligned vertically with the fluorescence excitation light source.

[0030] The droplet fusion device of this application has at least the following beneficial effects: In the droplet fusion device of this application, a laser detection unit and an image acquisition unit are used to achieve quality control during droplet generation and fusion. The laser detection unit is highly sensitive to changes in refractive index and has the advantage of fast response speed, thus enabling accurate detection of changes in refractive index 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 be cross-validated to ensure the accuracy of the detection. The physical information such as the size and velocity of the droplet can be mutually calibrated through joint detection, ensuring the accuracy of the droplet physical characteristic data, thereby enabling more accurate judgment of abnormalities in the droplet generation and fusion process. Thus, the droplet generation and fusion process can be monitored in real time with precision, and the quality control can be performed in real time by adjusting the parameters through the controller, significantly improving the reliability and efficiency of single-cell sequencing technology. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the droplet flow direction in the microfluidic chip of a droplet fusion device according to an embodiment of this application;

[0032] Figure 2 for Figure 1 A schematic diagram of droplet fusion in a microfluidic chip;

[0033] Figure 3 This is a schematic diagram of the frame structure of a droplet fusion device according to an embodiment of this application;

[0034] Figure 4 for Figure 3 A schematic diagram of the microfluidic chip structure in the droplet fusion device;

[0035] Figure 5 This is a three-dimensional structural diagram of a droplet fusion device according to an embodiment of this application;

[0036] Figure 6 for Figure 5Top view structural schematic of droplet fusion device in the application after removing three adjusting mechanisms of image acquisition system;

[0037] Figure 7 For Figure 6 Left view schematic of the application.

[0038] The element numbers in the figure are as follows:

[0039] First laser detection unit 100 (wherein, first infrared laser emitter 110, first infrared receiver 120);

[0040] Second laser detection unit 200 (wherein, second infrared laser emitter 210, second infrared receiver 220);

[0041] Third laser detection unit 300 (wherein, third infrared laser emitter 310, third infrared receiver 320);

[0042] Image acquisition unit 400 (wherein, camera 410, first adjusting mechanism 420, second adjusting mechanism 430, third adjusting mechanism 440);

[0043] Fluorescence detection unit 500 (wherein, fluorescence excitation light source 510, fluorescence receiving module 520);

[0044] First reflecting element 610, dichroic mirror 620, second reflecting element 630;

[0045] Gas supply assembly 700;

[0046] Controller 800;

[0047] Microfluidic chip 900 (wherein, first microfluidic channel A, second microfluidic channel B, fusion microfluidic channel C). DETAILED DESCRIPTION

[0048] The technical solutions of the present application are further described in detail below in combination with the accompanying drawings and specific embodiments.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of implementations of the present application. As used in this description, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0050] In the description of the present application, it needs to be understood that the terms "center", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0051] In the description of the present application, it needs to be understood that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be directly connected, or indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0052] Please refer to Figure 1 and Figure 2 The Y-shaped microfluidic chip 900 used in the droplet fusion device of the present application includes a first microfluidic channel A, a second microfluidic channel B and a fusion microfluidic channel C. Among them, the first microfluidic channel A and the second microfluidic channel B introduce different liquid flows respectively, and the liquid droplets generated after flowing in the microchannel; the first microfluidic channel A and the second microfluidic channel B meet at the fusion microfluidic channel C, and the liquid droplets generated in the first microfluidic channel A and the liquid droplets generated in the second microfluidic channel B realize fusion in the fusion microfluidic channel C, and the fusion process is shown in Figure 2 .

[0053] On the microfluidic chip 900, the first microfluidic channel A and the second microfluidic channel B are respectively provided with a first laser detection area and a second laser detection area. The image acquisition area is arranged at the intersection of the first microfluidic channel A, the second microfluidic channel B and the fusion microfluidic channel C. The fusion microfluidic channel C is responsible for droplet fusion and subsequent detection, and the fusion microfluidic channel C is provided with a third laser detection area and a fluorescence detection area, which ensures that the droplets still maintain high quality after fusion, and accurately detects the contents. The design of the fusion microfluidic channel C ensures that the droplets can smoothly pass through the subsequent detection area (third laser detection area and fluorescence detection area) after fusion.

[0054] Please refer to Figure 3 and Figure 4The droplet fusion device of the embodiment of the present application further comprises 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, a gas supply assembly 700 and a controller 800, and 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 gas supply assembly 700 are all signal connected to the controller 800 for centralized and unified control by the controller 800.

[0055] The first laser detection unit 100, the second laser detection unit 200 and the third laser detection unit 300 are respectively used for laser detection of the droplets in the first microfluidic channel A, the second microfluidic channel B and the fusion microfluidic channel C by laser, for monitoring the physical properties of the droplets, and for providing high-precision droplet size, speed and internal particle information through laser detection; the image acquisition unit 400 is used for real-time image acquisition of the fusion channel C, for ensuring the generation and fusion process of the droplets to be in a controllable state by analyzing the size, spacing and speed of the droplets in the acquired images, and for identifying abnormal conditions in the microfluidic channel to realize real-time detection; the fluorescence detection unit 500 is used for fluorescence detection of the droplets in the fusion channel C, for judging whether the droplets contain target substances (such as cells, nucleic acids, etc.) to be captured. The first microfluidic channel A and the second microfluidic channel B generate droplets by supplying gas inward through the gas supply assembly 700, and the generation speed and size of the droplets in the first microfluidic channel A and the second microfluidic channel B can be controlled by adjusting the gas pressure provided by the gas supply assembly 700 to the microchannels through the controller 800.

[0056] The laser detection utilizes the propagation characteristics of light in different media to convert the measured physical quantity into an optical signal, and then utilizes a photoelectric converter to convert the optical signal into an electrical signal, and after corresponding processing, corresponding measurement information is obtained. The specific working principle is as follows: due to the different propagation characteristics of light in different media, when the droplets pass through the microfluidic channel, the intensity of the optical signal will change, and the frequency and amplitude changes of the optical signal can reflect the generation frequency, size and spacing of the droplets; the infrared receiver captures these changes and converts them into an electrical signal, which is transmitted to the controller 800; the controller 800 receives the digital signal output from the laser detection unit, analyzes the signal by using a preset algorithm, and calculates the generation frequency of the droplets by the frequency and amplitude changes of the signal.

[0057] Specifically, 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 a 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 a 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 a third feedback light to generate a third laser detection signal.

[0058] 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), including a special wavelength LED (usually a near-infrared LED), an LED constant current source circuit, a focusing lens, a collimating lens, and an optical 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, an avalanche diode, a silicon photomultiplier, etc.), a signal processing circuit, etc.

[0059] 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 determine the size, speed, and whether the droplet contains particulate matter (such as a gel ball) by the laser waveform 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 individual microchannels can ensure that the droplets are uniform and of high quality before entering the fusion microchannel C, providing a reliable basis for subsequent fusion operations. By monitoring the droplet generation frequency of the first microchannel A and the second microchannel B in real time through the first laser detection unit 100 and the second laser detection unit 200, and adjusting the gas supply pressure of the gas supply assembly 700 through 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 frequencies of the two microchannels maintain a 1:1 ratio, creating good prerequisites for subsequent fusion in the fusion microchannel C.

[0060] The third laser detection unit 300 is used to detect the physical characteristics of the droplets in the fusion microchannel C after fusion. When the droplets pass through the third laser detection area of the fusion microchannel C, the controller 800 judges whether the droplets in the third laser detection area are successfully fused after receiving the third laser detection signal of the third infrared receiver 320, and ensures that the droplet size, speed and morphology meet the experimental requirements through waveform analysis. The third laser detection unit 300 on the fusion microchannel C plays a role of connecting the previous and the next, ensuring that the droplets entering the fluorescence detection area of the subsequent fluorescence detection module 500 are complete and reliable.

[0061] The image acquisition unit 400 uses visual recognition technology to capture images during droplet generation and electrical fusion through the camera 410, and analyzes the characteristics of the droplets using image processing algorithms. The specific working principle is as follows: the images of the droplet generation and electrical fusion process captured by the camera 410 in real time 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 electrical fusion efficiency of the droplets through edge detection, image segmentation and feature extraction algorithms; according to the preset standard, the controller 800 judges whether there is an abnormality in the droplet generation and electrical fusion process. For example, whether the droplet shape is regular, the generation frequency is consistent, and the fusion efficiency meets the standard. The controller 800 can identify the problems that may occur in the droplet generation and electrical fusion process through the image information provided by the image acquisition unit 400, and feedback in time, for example, by displaying on the display device (not shown in the figure) and issuing a reminder.

[0062] The camera 410 is installed below the droplet generation area of the first microchannel A and 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 410 uses a high-frame-rate camera that can capture fast-moving droplets in the image acquisition area. The collimating lens and the fill light can also be provided in the image acquisition unit 400 in cooperation with the camera 410, the collimating lens is used to focus light, and the fill light provides sufficient lighting conditions, so that clear images can be obtained in low light environments.

[0063] The image acquisition unit 400 comprises, in addition to the camera 410, a first adjusting mechanism 420, a second adjusting mechanism 430 and a third adjusting mechanism 440. The camera 410 is installed at the movable end of the first adjusting mechanism 420, the first adjusting mechanism 420 is installed at the movable end of the second adjusting mechanism 430, and the second adjusting mechanism 430 is installed at the movable end of the third adjusting mechanism 440. The first adjusting mechanism 420, the second adjusting mechanism 430 and the third adjusting mechanism 440 can move the movable end in three different directions respectively, so that the position of the camera 410 can be adjusted in three directions, the shooting area and the shooting distance of the camera 410 are adjusted, and a larger shooting range can be achieved within a certain range. In the illustrated embodiment, the moving directions of the movable end of the first adjusting mechanism 420, the movable end of the second adjusting mechanism 430 and the movable end of the third adjusting mechanism 440 are perpendicular to each other, i.e. arranged along the Z direction (up-down direction), the Y direction (front-rear direction) and the X direction (left-right direction) respectively, to realize three-dimensional adjustment.

[0064] The camera 410 in the image acquisition unit 400 can adopt a large-area CCD module to capture the images of the liquid drops in the first micro-channel A, the second micro-channel B and the fusion micro-channel C in real time. The controller 800 analyzes the size, spacing and speed of the liquid drops and other indicators to assist other detection modules to ensure that the generation and fusion process of the liquid drops are in a controllable state. The image acquisition unit 400 can also identify abnormal conditions in the micro-channels, such as bubbles, foreign matter, liquid drop jamming or pipeline blockage, etc. Since these problems are difficult to be detected by the laser detection module or the fluorescence detection module, the image analysis of the image acquisition unit 400 becomes an indispensable monitoring means to provide more comprehensive protection for experimental operation. Therefore, the image analysis of the image acquisition unit 400 can further verify the morphology and flow state of the liquid drops and find abnormal problems that cannot be identified by other detection means.

[0065] The fluorescence detection unit 500 is used to detect whether the target substance to be captured is contained in the fused liquid drop, which comprises a fluorescence excitation light source 510 and a fluorescence receiving module 520. The fluorescence excitation light source 510 is arranged at one side of the fusion micro-channel C and emits fluorescence excitation light, and the fluorescence receiving module 520 is installed at the other side and receives the excited fluorescence to generate a fluorescence detection signal. In the experimental process, the target cells or nucleic acids are labeled in advance by fluorescent dyes. When these markers enter the liquid drop and flow through the fluorescence detection area of the fusion micro-channel C, the fluorescence excitation light source 510 emits fluorescence excitation light to irradiate the liquid drop in the fluorescence detection area, and whether the target substance is contained in the liquid drop is judged according to the fluorescence received by the fluorescence receiving module 520. The strength of the fluorescence received by the fluorescence receiving module 520 can directly reflect the concentration or quantity of the target substance in the liquid drop.

[0066] Due to the small size of the microfluidic chip 900, the first microfluidic channel A, the second microfluidic channel B, and the fusion microfluidic channel C are small in spacing. Therefore, how to arrange 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 in the limited space around the microfluidic chip 900 without interference between each other is also a key point to be solved.

[0067] For further reference, please see Figure 5 , Figure 6 and Figure 7 The microfluidic chip 900 is horizontally placed on the plane in the X direction and the Y direction, and each component of 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 is arranged around the microfluidic chip 900.

[0068] 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 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 fluorescence excitation light are all irradiated from above to each microfluidic channel 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 arranged below the microfluidic chip 900 (lower than the microfluidic chip 900 in the Z direction).

[0069] More specifically, the first infrared laser emitter 110 and the second infrared laser emitter 310 are arranged opposite to each other in the front-rear direction and emit the first detection laser and the second detection laser along the Y direction, respectively. To achieve the emitted detection laser irradiating onto the microfluidic chip 900 along the Z direction, a first reflecting element 610 is arranged above the first laser detection area of the first microfluidic channel A and the second laser detection area of the second microfluidic channel B. The first reflecting element 610 reflects the first detection laser and the second detection laser from the Y direction on both sides to the light beams downward along the Z direction. The first reflecting element 610 has a first reflecting surface that reflects the first detection laser emitted by the first infrared laser emitter 110 along the Y direction to the first microfluidic channel A along the Z direction. The first reflecting element 610 has a second reflecting surface that reflects the second detection laser emitted by the second infrared laser emitter 110 along the Y direction to the second microfluidic channel B along the Z direction. In the illustrated embodiment, the first reflecting element 610 can be a prism, which is composed of a prism with a first reflecting surface and a prism with a second reflecting surface.

[0070] More specifically, the third infrared laser emitter 310 is arranged directly above the third laser detection region of the fusion microfluidic channel C.

[0071] As described above, the camera 410 is arranged below the image acquisition region at the intersection of the first microfluidic channel A, the second microfluidic channel B and the fusion microfluidic channel C, and is movable 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 arranged below the microfluidic chip 900 need to be arranged to avoid the position below the image acquisition region. Based on this, a dichroic mirror 620 is arranged between the image acquisition region of the microfluidic chip 900 and the camera 410, the dichroic mirror 620 is arranged at an angle of 45° with the Z direction, and 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 after the first detection laser, the second detection laser and the third detection laser along the Z direction to the front along the Y direction, and transmits the white light along the Z direction, so that the camera 410 can perform image acquisition on the image acquisition region.

[0072] 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, so the third infrared receiver 320 is arranged in the Y direction to face the reflecting surface of the dichroic mirror 620, and the first infrared receiver 120 and the second infrared receiver 220 are arranged on the two sides of the X direction respectively. In the Y direction of the reflecting surface of the dichroic mirror 620, a second reflecting member 630 is arranged, which reflects the first feedback light and the second feedback light from the dichroic mirror 620 along the Y direction to propagate to the two sides along the X direction. The second reflecting member 630 has a third reflecting surface, which reflects the light reflected by the first detection laser emitted by the first infrared laser emitter 110 through the first reflecting member 610, through the first microfluidic channel A and through 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 reflecting member 630 has a fourth reflecting surface, which reflects the light reflected by the second detection laser emitted by the second infrared laser emitter 210 through the first reflecting member 610, through the first microfluidic channel B and through 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 reflecting member 630 can be a prism, which is composed of a prism having the third reflecting surface and a prism having the fourth reflecting surface.

[0073] More specifically, the fluorescent excitation light source 510 is arranged directly above the fluorescent detection region of the fusion microfluidic channel C, the fluorescent receiving module 520 is arranged directly below the fluorescent detection region, and the fluorescent receiving module 520 is aligned with the fluorescent excitation light source 510 in the Z direction.

[0074] The droplet fusion device of the present application is provided with a laser detection unit (first laser detection unit 100, second laser detection unit 200 and third laser detection unit 300) and a fluorescence detection unit 500, and simultaneously uses image analysis technology of the image acquisition unit 400 to realize global real-time monitoring. Through the cooperative work of different detection means, the accuracy and stability of the droplet operation in the experiment process are ensured, and the experimental parameters can be automatically adjusted to optimize the experimental conditions. The droplet fusion device 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 change of the optical signal is captured by the infrared receiver and transmitted to the processor 800. The processor 800 analyzes the frequency change of the optical signal and calculates the droplet generation frequency and the droplet size of the first microchannel A and the second microchannel B. According to the analysis result 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 assembly 700, so as to ensure the stability of the droplet generation of the first microchannel A and the second microchannel B and the frequency to maintain a ratio of 1:1. This real-time and high-precision detection and adjustment method can ensure that the droplets reach the best state before electric fusion.

[0075] The droplet fusion device of the present application can monitor the fusion process: the image acquisition unit 400 takes real-time images of the droplets in the electric fusion area (image acquisition area), and the controller 800 detects the shape, size and fusion condition of the droplets through image analysis algorithm, and identifies the shape of the fused droplets in real time, analyzes the efficiency and success rate of electric fusion. Specifically, the image acquisition unit 400 takes real-time images of the droplets in the first microchannel A and the second microchannel B, and the controller 800 extracts the contour and generation frequency of the droplets through edge detection and image segmentation. When the droplet generation frequency and the 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 gas supply assembly 700 until the preset value is met.

[0076] In addition, after the droplet generation frequency data provided by the first laser detection unit 100 and the second laser detection unit 200 and the fusion image data provided by the image acquisition unit 400 are integrated in the controller 800, the droplet generation frequency and the fusion efficiency are comprehensively analyzed, and it is confirmed 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 assembly 700 to ensure that the frequency of the two microchannels maintains a ratio of 1:1, thereby further improving the accuracy of monitoring and adjustment.

[0077] When the controller 800 analyzes that the droplet generation frequency is inconsistent or the fusion efficiency is low, a pre-warning prompt will be issued immediately, and the relevant images and parameters will be displayed on the display device. Maintenance personnel can adjust the related parameters of droplet generation and fusion, such as flow rate, pressure and electric field strength, according to the prompt of the display device. The display device displays the adjusted related parameters to show the adjustment effect, ensuring that the droplet generation frequency and fusion efficiency reach the best state.

[0078] In the droplet fusion device of the present application, laser detection unit, image acquisition unit and fluorescence detection unit are used to realize quality control during droplet generation and fusion. Not only the monitoring and adjustment of droplet generation frequency and fusion efficiency, but also the complementary advantages of multiple technologies are as follows:

[0079] (1) High sensitivity of laser detection: The laser detection unit is very sensitive to the change of refractive index, especially when the droplet encapsulates the DNA special label gel ball. Because the material of the gel ball (such as hydrogel) is close to transparent, image method may not be able to effectively identify the DNA label gel ball encapsulated in the water-in-oil structure, which may lead to misjudgment. Laser detection can accurately detect the change of refractive index inside the droplet, so as to accurately identify the encapsulated DNA label gel ball.

[0080] (2) High response speed of laser detection: The fastest response speed of laser detection technology can reach picosecond level (10 -12 -9 seconds), and the response speed of visual recognition technology with general industrial camera (small size camera) as detection device is about millisecond level (10 -3 -3 seconds). Laser detection technology makes up for the problem of insufficient high-speed droplet response speed under low cost and small space.

[0081] (3) Intuitiveness of image acquisition: Image acquisition unit can detect the shape, size and fusion condition of droplet through image analysis, which is very important for detecting the physical state of droplet and the morphological change after fusion.

[0082] (4) Data fusion and comprehensive analysis: The droplet generation frequency and internal structure data provided by laser detection unit are combined with the droplet morphology and fusion efficiency data provided by image acquisition unit, and the controller performs comprehensive analysis. The monitoring data of laser detection unit and the monitoring data of image acquisition unit can be verified through cross-validation data to ensure the accuracy of detection. The physical information such as size and speed of droplet can be calibrated through common detection, ensuring the accuracy of droplet physical property data, so as to more accurately judge the abnormal situation in the process of droplet generation and fusion, and provide timely adjustment suggestions. In addition, the detection data of fluorescence detection unit can further confirm whether the droplet contains target substances by combining with physical data, so as to reduce false positives.

[0083] In summary, the application provides a droplet fusion device, which combines a laser detection unit, an image acquisition unit and a fluorescence detection unit, and proposes a multi-level quality control scheme to realize real-time and accurate monitoring of the droplet generation and fusion process, and can realize real-time and automatic quality control by adjusting parameters, thereby significantly improving the reliability and efficiency of single-cell sequencing technology.

[0084] It should be noted that in this paper, the term "includes", "contains" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or device. Without more limitations, the element defined by the statement "includes a" does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0085] The above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the application, which should be covered by the protection scope of the application. Therefore, the protection scope of the application should be subject to the protection scope of the claims.

Claims

1. A droplet fusion apparatus characterized by comprising: The application relates to a microfluidic chip, a first microfluidic channel, a second microfluidic channel and a fusion microfluidic channel, wherein the first microfluidic channel and the second microfluidic channel converge into the fusion microfluidic channel, a first laser detection area and a second laser detection area are respectively formed on the first microfluidic channel and the second microfluidic channel, and an image acquisition area is formed at the intersection of the first microfluidic channel, the second microfluidic channel and the fusion microfluidic channel. A first laser detection unit is used for emitting a first detection laser, receiving a first feedback light formed after passing through the first laser detection area and generating a first laser detection signal. A second laser detection unit is 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 is used for shooting an image of the image acquisition area. A fluorescence detection unit is used for emitting a fluorescence excitation light, receiving a fluorescence excited after passing through a fluorescence detection area on the fusion microfluidic channel and generating a fluorescence detection signal. A controller receives detection signals of the first laser detection unit and the second laser detection unit and image information of the image acquisition unit and adjusts the speed and pressure of liquid flow in the first microfluidic channel and the second microfluidic channel according to the detection signals and the image information. The first laser detection unit comprises a first infrared laser emitter and a first infrared receiver; the second laser detection unit comprises a second infrared laser emitter and a second infrared receiver; the image acquisition unit comprises a camera device, and the camera device is below the image acquisition area; and the fluorescence detection unit comprises a fluorescence excitation light source and a fluorescence receiving module.

2. The droplet fusion apparatus according to claim 1, wherein, The first infrared laser emitter, the second infrared laser emitter and the fluorescence excitation light source are arranged above the microfluidic chip, and the first infrared receiver, the second infrared receiver and the fluorescence receiving module are arranged below the microfluidic chip. The image acquisition unit further comprises a first adjusting mechanism, a second adjusting mechanism and a third adjusting mechanism, the camera device is installed on the movable end of the first adjusting mechanism, the first adjusting mechanism is installed on the movable end of the second adjusting mechanism, the second adjusting mechanism is installed on the movable end of the third adjusting mechanism, and the moving directions of the movable ends of the first adjusting mechanism, the second adjusting mechanism and the third adjusting mechanism are perpendicular to each other.

3. The droplet fusion apparatus according to claim 2, wherein, The application further relates to a third laser detection unit which is used for emitting a third laser signal, receiving a third feedback light formed after passing through a third laser detection area on the fusion microfluidic channel and generating a third laser detection signal.

4. The droplet fusion apparatus according to claim 3, wherein The third laser detection unit comprises a third infrared laser emitter and a third infrared receiver; the third infrared laser emitter is arranged above the microfluidic chip, and the third infrared receiver is arranged below the microfluidic chip. A first reflector 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 oppositely arranged on the two sides of the first reflector in the front-rear direction, and the first reflector reflects the first detection laser and the second detection laser downward.

5. The droplet fusion apparatus according to claim 4, characterized by: ​ 6. The droplet fusion apparatus according to claim 5, wherein: ​ 7. The droplet fusion apparatus according to claim 6, characterized by: The third infrared laser emitter is arranged directly above the third laser detection area.

8. The droplet fusion apparatus according to claim 7, characterized by: A dichroic mirror is arranged between the image acquisition area and the camera, the dichroic mirror transmits white light and reflects the downward first feedback light, the second feedback light and the third feedback light forward.

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

10. The droplet fusion apparatus of claim 2, wherein: The fluorescent excitation light source is arranged directly above the fluorescent detection area, and the fluorescent receiving module is aligned with the fluorescent excitation light source in the up-down direction.

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