Gas supply assembly, single cell sequencing library building equipment and control method thereof

Abstract

The invention provides a gas supply assembly, a single cell sequencing library building device and a control method thereof.The gas supply assembly is used for providing gas for a chip to form micro-droplets and sequentially comprises a gas generator, a gas generator pressure sensor, a regulation and control valve and a pressure output module in the gas flowing direction; the pressure output module supplies gas to the reaction hole of the chip, and the outlet side of the pressure output module is provided with a chip pressure sensor used for detecting airway pressure. According to the air supply assembly and the single cell sequencing library building equipment, the chip pressure sensor arranged on the outlet side of the pressure output module is used for monitoring the pressure change in the experiment process in real time, so that the pressure and the pressure change can be monitored in real time at the position closest to the chip, and abnormity such as hole blockage or unstable air pressure can be found in time; therefore, the user is allowed to adjust or stop the experiment in real time, the situation that faults occur without prompt is avoided as much as possible, and the reliability and success rate of the experiment are remarkably improved through the real-time feedback mechanism.

Description

Technical Field

[0001] The present application relates to the technical field of sequencers, and in particular to a gas supply component, a single-cell sequencing library construction device, and a control method thereof. Background Art

[0002] At present, single-cell sequencing technology is mainly divided into two categories: droplet-based single-cell sequencing and plate-based single-cell sequencing. Among them, droplet-based single-cell sequencing is a high-throughput technology widely used in the industry. This technology processes single cells and reaction reagents by encapsulating them in tiny oily droplets. These droplets act independently as reaction chambers and can process thousands to tens of thousands of cells in parallel, which is dozens of times the processing capacity of the microplate method. Each droplet usually contains a unique DNA molecule with a barcode, which is used to identify RNA or DNA molecules derived from the same cell in subsequent genome sequencing. The main advantages of this technology are its high throughput and low sample input requirements.

[0003] Nevertheless, the droplet method has obvious limitations in actual operation. In the prior art, the pressure sensor used to detect the failure of the gas supply component is installed at the rear end of the control valve. When the failure of the gas supply component occurs at the rear end of the pressure sensor to the chip input port, the pressure sensor cannot sense the failure. Therefore, the pressure display is normal even if the failure occurs, resulting in the experiment continuing even if it fails, wasting specimens and reagents. Summary of the invention

[0004] In order to solve the existing technical problems, the present application provides a gas supply component capable of detecting the experimental pressure in real time, a single-cell sequencing library construction device and a control method thereof, which can avoid the situation where a fault occurs but no prompt is given.

[0005] To achieve the above purpose, the technical solution of the embodiment of the present application is implemented as follows:

[0006] On the one hand, the embodiment of the present application provides a gas supply component for supplying gas to a chip to form droplets, wherein the gas supply component includes a gas generator, a gas generator pressure sensor, a control valve and a pressure output module in sequence along the gas flow direction, wherein the pressure output module supplies gas to the reaction hole of the chip, and a chip pressure sensor for detecting airway pressure is arranged at the outlet side of the pressure output module;

[0007] Corresponding to the number n of reaction holes on each chip, n air tributaries are provided from the gas generator to the pressure output module, one regulating valve is provided on each of the n air tributaries, n air supply passages are provided in the pressure output module corresponding to the n air tributaries, and one pressure sensor is provided on the outlet side of each air supply passage, wherein n is an integer greater than or equal to 1; the regulating valve adjusts the air supply pressure of the air tributary according to the pressure detection data of the chip pressure sensor on the same air tributary;

[0008] The gas supply assembly is also provided with a second gas generator, and the gas generator and the second gas generator are connected to the gas path leading to the control valve through a first three-way valve, so that the gas generator and the second gas generator are connected to the control valve in one of two ways.

[0009] In one embodiment, in the pressure output module, m gas supply interfaces are arranged in series on each gas supply passage corresponding to m chips, and the same reaction holes on the m chips are connected to the m gas supply interfaces connected in series on the same gas supply passage, wherein m is an integer greater than or equal to 1.

[0010] In one of the embodiments, the gas supply assembly is further provided with a control valve pressure sensor, and the control valve pressure sensor is connected to the gas path from each of the control valves to the pressure output module.

[0011] In one embodiment, an n+1th airway is further provided between the gas generator and the pressure output module, and a second regulating valve is provided on the n+1th airway, and the second regulating valve is respectively connected to the airway where each of the regulating valves is located through a second three-way valve, so that the airway where any of the regulating valves is located can be switched to the airway where the second regulating valve is located through the corresponding second three-way valve and connected to the pressure output module.

[0012] On the other hand, an embodiment of the present application provides a single-cell sequencing library construction device, including a shell, a chip transport tray, a display module, a control main board, and the gas supply assembly described above, wherein the chip transport tray is used to carry the chip and can be moved outward and inward in the shell, the display module is arranged on the shell, and the control main board and the gas supply assembly are arranged in the shell; the display module, the control valve and the chip pressure sensor signal are connected to the control main board, and the control main board receives the pressure detection data from the chip pressure sensor and controls the control valve to adjust the gas supply pressure.

[0013] In one of the embodiments, the control mainboard sends the pressure detection data of the chip pressure sensor to the display module for display; the control mainboard sets a pressure change alarm threshold so that when the pressure detection data of the chip pressure sensor reaches the pressure change alarm threshold, an abnormality is prompted on the display module.

[0014] In one embodiment, the single-cell sequencing library construction device also includes a real-time image acquisition component disposed in the housing, and the real-time image acquisition component includes a camera module, and the camera module is disposed below the chip transport tray to photograph the chip on the chip transport tray from bottom to top.

[0015] On the other hand, an embodiment of the present application provides a control method for the single-cell sequencing library construction device as described above, comprising the following steps: when the pressure detection data of the chip pressure sensor is abnormal, prompting the user to terminate the experiment.

[0016] In one embodiment, the control method of the single-cell sequencing library construction device further includes the following steps:

[0017] When the pressure detection data of the gas generator pressure sensor is abnormal, the control main board controls the first three-way valve to switch to connect the second gas generator with the regulating valve, and the gas generator pressure sensor performs pressure detection again;

[0018] If the pressure detection data of the gas generator pressure sensor returns to normal, the experiment continues normally; otherwise, the user is prompted to terminate the experiment.

[0019] In one embodiment, the gas supply assembly is further provided with a second regulating valve, a regulating valve pressure sensor and a second regulating valve pressure sensor, the regulating valve pressure sensor is connected to the bronchial passage from each regulating valve to the pressure output module, and the second regulating valve and the second regulating valve pressure sensor are connected to the bronchial passage from each regulating valve to the pressure output module through a second three-way valve;

[0020] The control method of the single-cell sequencing library building device also includes the following steps:

[0021] When the pressure detection data of the gas generator pressure sensor is normal and the pressure detection data of a certain control valve pressure sensor is abnormal, the control mainboard controls the second three-way valve on the bronchial passage where the control valve pressure sensor is located to switch so that the bronchial passage where the second control valve is located is connected to the pressure output module, and the second control valve pressure sensor performs pressure detection;

[0022] If the pressure detection data of the second control valve pressure sensor is normal, the experiment continues normally; otherwise, the user is prompted to terminate the experiment.

[0023] The gas supply assembly, single-cell sequencing library construction device and control method thereof of the present application have at least the following beneficial effects: in the gas supply assembly and single-cell sequencing library construction device of the present application, the pressure change during the experiment is monitored in real time by the chip pressure sensor arranged at the outlet side of the pressure output module, so that the pressure and pressure change can be monitored in real time at the position closest to the chip and abnormalities such as hole blockage or unstable air pressure can be discovered in time, thereby allowing the user to adjust or terminate the experiment in real time, and avoiding the occurrence of failures without prompts as much as possible. This instant feedback mechanism significantly improves the reliability and success rate of the experiment;

[0024] The gas supply component and single-cell sequencing library construction equipment of the present application are equipped with a pressure sensor at each fault section. Through the integrated linkage of multiple pressure sensors, the pressure fault point can be accurately determined in real time. At the same time, a second gas generator and a second regulating valve are arranged at the pressure fault points such as the gas generator and the regulating valve. According to the pressure fault point located in the previous step, the spare second gas generator and the second pressure regulating valve can be started, thereby realizing automatic repair and elimination of the pressure fault. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the structure of a single-cell sequencing library building device in a first state according to an embodiment of the present application;

[0026] Figure 2 for Figure 1 A schematic diagram of the structure of the single-cell sequencing library construction device in the second state;

[0027] Figure 3 for Figure 1 Schematic diagram of the internal structure of the single-cell sequencing library construction device;

[0028] Figure 4 for Figure 3 A schematic diagram of the internal structure of the single-cell sequencing library construction device from another angle;

[0029] Figure 5 This is a schematic structural diagram of an air supply assembly according to an embodiment of the present application;

[0030] Figure 6 for Figure 5 A schematic structural diagram of the air supply assembly from another angle;

[0031] Figure 7 This is a schematic diagram of the connection structure framework between the gas supply assembly and the chip according to one embodiment of the present application;

[0032] Figure 8This is a schematic diagram of the connection structure framework between the gas supply assembly and the chip according to another embodiment of the present application;

[0033] Fig. 9 for Figure 3 A schematic diagram of the structure of the real-time image acquisition component in FIG.

[0034] Fig.10 for Fig. 9 A structural diagram of the real-time image acquisition component from another angle;

[0035] Fig.11 for Fig. 9 Schematic diagram of the structure of the real-time image acquisition component from an upward perspective.

[0036] The components in the figure are labeled as follows:

[0037] Housing 100;

[0038] Chip transport tray 200;

[0039] Display module 300;

[0040] Control main board 400;

[0041] Gas supply assembly 500 (including gas generator 510, second gas generator 520, partial pressure cylinder 511, gas generator pressure sensor 530; control valve 540, second control valve 550, control valve pressure sensor 560, second control valve pressure sensor 551; pressure output module 570; chip pressure sensor 580);

[0042] Real-time image acquisition component 600 (including camera module 610, optical lens 620, connector 630, light source lamp 640, light source lamp mounting seat 650, light source lamp adjustment driver 660);

[0043] Mounting frame 700 (including mounting base plate 710, mounting seat 720, mounting vertical plate 730, and adjusting slide rail 740);

[0044] Chip 800. DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Microfluidics refers to a technology that manipulates fluids in micrometer-scale space. This technology can miniaturize the basic functions of chemistry, biology and other laboratories onto a chip of a few square centimeters, so it is also called a chip laboratory (Lab-on-a-chip). Microfluidic chip systems can manipulate the flow of fluids in tiny channels or components ranging from tens to hundreds of micrometers, and the volume of fluids manipulated can be as small as 10 -18 ~10 -9 L. It integrates the basic operations of biochemical experiments such as sample reaction, preparation, separation, and detection into a very small chip, and uses controllable fluids to form a network of microchannels throughout the microfluidic system. While realizing the functions of conventional biochemical laboratories, it reduces the cost of analysis and detection, speeds up the reaction speed, improves the reaction efficiency, and makes the experiment more controllable.

[0050] The generation of droplets on a microfluidic chip is a process in which one phase of fluid is dispersed in another phase of immiscible or partially miscible fluid. One of the two immiscible liquids is used as the continuous phase and the other as the dispersed phase. The dispersed phase is dispersed in a tiny volume (10 -15 ~10 -9 L) units are dispersed in the continuous phase to form droplets.

[0051] Foreign matter may sometimes exist in a single microchannel during droplet generation. These foreign matter may cause blockage, or even if they do not block, they may affect the flow rate. The presence of foreign matter will affect the flow rate of the solution in a single microchannel, which may easily lead to uneven droplet sizes or discontinuous droplet intervals due to too slow a flow rate. Sometimes, there will be pressure instability in a single microchannel, which will affect the flow rate of the solution, causing uneven droplet sizes and discontinuous intervals. The existing technology cannot monitor and control these abnormal phenomena in real time, and cannot control the quality of the generated droplets in real time, resulting in abnormal droplets entering the droplet storage chamber and mixing with normal droplets. If the number of abnormal droplets is large, they can be found under a microscope by size differences, but even if the abnormality is found, it cannot be remedied and the experiment can only be repeated, resulting in waste of specimens, especially precious trace samples; at the same time, the reagents and consumables for single-cell sequencing are also wasted, resulting in cost and time waste. If the number of abnormal droplets is small or the operator does not observe carefully, it is difficult to distinguish and count abnormal droplets under a microscope, and these droplets will continue to the next step of amplification and library construction, resulting in erroneous results, which seriously affects scientific research and clinical diagnosis and treatment. In the above two cases, it is only possible to observe and discover under a microscope after the experiment is over. The existing technology cannot perform real-time quality control on the quality of the droplets before the experiment is over, and it is even more impossible to intervene in the quality of abnormal droplets during the experiment.

[0052] The single-cell sequencing library preparation device of the present application uses droplet microfluidics technology to complete the capture, separation, and labeling of single cells in one package. It can be used in conjunction with a single-cell sequencing kit to achieve flexible, stable, and efficient single-cell sequencing library preparation and other tasks.

[0053] See also Figure 1 and Figure 2 The single-cell sequencing library building device of one embodiment of the present application includes a housing 100, a chip transport tray 200, a display module 300, a control mainboard 400, an air supply component 500 and a real-time image acquisition component 600 (see Figures 3 to 8 ). Among them, the chip transport tray 200 is used to carry the chip 800, and can be relatively movably arranged in the housing 100 through a moving mechanism, and can be moved outward and inward relative to the housing 100 to transport the chip 800; the display module 300 is arranged on the housing 100, and is used to display relevant information to the user and / or provide an operable interface for the user; the control main board 400, the air supply component 500 and the real-time image acquisition component 600 are all fixedly installed in the housing 100 through the mounting frame 700. The single-cell sequencing library construction device defines mutually perpendicular X-direction, Y-direction and Z-direction. In the illustrated embodiment, the X-direction and the Y-direction are horizontal directions, and the Z-direction is a vertical direction.

[0054] The housing 100 can be enclosed by a plurality of plates to form a space for accommodating components such as the control mainboard 400, the air supply component 500 and the real-time image acquisition component 600. In the illustrated embodiment, the front end of the housing 100 is an opening tilted upward, so that the screen of the display module 300 can be installed at the opening in an upward tilted manner. The tilt angle of the screen conforms to the user's viewing habits and is convenient for viewing and / or operation. The display module 300 can use a touch screen, or an operation button can be provided next to the screen, so that the user can input instructions through the touch screen or the operation button. That is, the display module 300 can have both display function and input function.

[0055] The single-cell sequencing library construction device has two states: Figure 1 and Figure 2 As shown in Figure 1 In the first state shown, the chip transport tray 200 is retracted into the housing 100, and the single-cell sequencing library building device can work; Figure 2 In the second state shown, the chip transport tray 200 extends forward from the housing 100 along the Y direction so that the chip 800 can be placed on or removed from the chip transport tray 200. The chip transport tray 200 is used to clamp the chip 800, and at least one chip 800 can be loaded thereon. In the illustrated embodiment, the chip transport tray 200 is disposed below the display module 300, and four chips 800 can be placed side by side along the X direction thereon; each chip 800 has four reaction holes.

[0056] The control mainboard 400 is the centralized control core of the entire single-cell sequencing library building device, responsible for coordinating and managing the functions of each component, and completing the single-cell sequencing library building through the coordinated work of each component controlled by the control mainboard 400. For example, the display module 300, the gas supply component 500, and the real-time image acquisition component 600 are all signal-connected and controlled by the control mainboard 400.

[0057] The single-cell sequencing library building device uses airflow to drive the fluid to move, so that the fluid forms micro-droplets. The gas supply component 500 is used to provide gas to the chip 800 on the chip transport tray 200 to form micro-droplets.

[0058] Please refer to Figures 3 to 7The gas supply assembly 500 of one embodiment of the present application includes a gas generator 510, a control valve 540 and a pressure output module 570 in sequence along the gas flow direction. After the gas passes through the gas generator 510 and the control valve 540 in sequence, the pressure output module 570 supplies gas to the reaction hole of the chip 800. The gas generator 510 and the control valve 540 are both connected to the control main board 400 by signals and controlled by the control main board 400. The pressure output module 570 is correspondingly arranged above the chip transport tray 200 along the Z direction. When the chip transport tray 200 retracts into the housing 100 along the Y direction, the gas supply interface at the lower end of the pressure output module 570 is connected to the reaction hole of the chip 800 in the Z direction.

[0059] In order to further improve the gas supply quality of the gas supply assembly, a gas impurity removal module (not shown) can be provided at the front end of the gas generator 510. The gas impurity removal module is used to remove moisture or other impurities in the gas. Removing moisture from the gas can prevent water vapor from affecting the experimental accuracy and equipment performance. Dry gas helps maintain the stability of the system and extend the service life of the equipment.

[0060] The gas generator 510 provides stable and adjustable gas pressure for use in the entire single-cell sequencing library construction experiment process. The gas generator 510 is a key device for maintaining the pressure environment required for the experiment and is the basis for ensuring that the experimental conditions meet the preset standards.

[0061] At the same time, the gas supply assembly 500 is also provided with a second gas generator 520, and the gas generator 510 and the second gas generator 520 are connected to the front end of the gas path leading to the control valve 540 through a first three-way valve (not shown). The second gas generator 520 signal is connected to the control main board 400, and the control main board 400 controls the gas generator 510 and the second gas generator 520 to generate a gas source in the gas path leading to the control valve 540 in a selective manner. That is, the second gas generator 520 can be used as a spare part of the gas generator 510, so that when it is determined that the gas generator 510 is abnormal, the gas supply assembly 500 switches from the gas generator 510 providing the gas source to the control valve 540 to the second gas generator 520 providing the gas source to the control valve 540.

[0062] Corresponding to each chip 800 , a plurality of reaction holes are provided. The gas source generated by the gas generator 510 and the second gas generator 520 is divided into a plurality of branch airways from a main airway by the pressure-dividing cylinder 511 and then leads to the regulating valve 540 .

[0063] In order to more accurately determine whether the gas generator 510 is abnormal, a gas generator pressure sensor 530 is connected to the rear end of the gas generator 510 and the second gas generator 520 (i.e., the gas path from the first three-way valve to the control valve 540). In the illustrated embodiment, the gas generator pressure sensor 530 is connected to the pressure-dividing cylinder 511. The gas generator pressure sensor 530 is used to monitor the pressure at the current position in real time, and the gas generator pressure sensor 530 signal is connected to the control main board 400 to send the pressure detection data to the control main board 400.

[0064] More specifically, in the initial state, the first three-way valve connects the gas generator 510 with the pressure-dividing cylinder 511 at the rear end. When the gas generator pressure sensor 530 at the rear end of the gas generator 510 detects abnormal pressure data, it is determined that the gas generator 510 has failed. At this time, the control motherboard 400 controls the first three-way valve to automatically switch to the spare second gas generator 520, and the gas generator pressure sensor 530 performs pressure detection again. If the pressure of the gas generator pressure sensor 530 returns to normal, the experiment continues normally, otherwise the user is prompted to terminate the experiment in time. The control valve 540 is used to control the rate and amount of gas flowing into each reaction hole of the chip 800 to ensure the uniformity and stability of the experimental conditions. The control valve 540 can be a proportional pressure valve.

[0065] Corresponding to the number n (n≥1, and is an integer) of reaction holes on each chip 800, the main airway from the gas generator 510 to the chip 800 is divided into n+1 branches by the pressure-dividing cylinder 511, wherein: a regulating valve 540 and a regulating valve pressure sensor 560 are provided on each of the n branches, and are connected to the pressure output module 570; a second regulating valve 550 and a second regulating valve pressure sensor 551 are provided on the n+1 branch, and are bypassed to the remaining n branches. The pressure output module 570 is correspondingly provided with n gas supply passages, and a chip pressure sensor 580 is provided on the outlet side of each gas supply passage (branch).

[0066] On the n+1th branch airway, the second control valve 550 and the second control valve pressure sensor 551 are respectively connected to the remaining n branches to the front end leading to the pressure output module 570 through n second three-way valves (not shown). The second control valve 550 signal is connected to the control main board 400, and the control main board 400 controls one of the control valves 540 and the second control valve 550 to selectively control the flow rate and flow volume of the gas in the air path leading to the pressure output module 570. That is, the second control valve 550 can be used as a spare part of the control valve 540, so that when it is determined that a certain control valve 540 is abnormal, the gas supply component 500 switches from the control valve 540 to the corresponding gas supply path of the pressure output module 570 to the second control valve 550 to provide the gas source to the corresponding gas supply path of the pressure output module 570.

[0067] In order to more accurately determine whether the control valve 540 is abnormal, a control valve pressure sensor 560 is connected to the rear end of each control valve 540. The control valve pressure sensor 560 is used to monitor the pressure at the current position in real time, and the control valve pressure sensor 560 signal is connected to the control main board 400 to send the pressure detection data to the control main board 400.

[0068] More specifically, when the control valve pressure sensor 560 at the rear end of a certain control valve 540 detects abnormal pressure data, it is determined that the control valve 540 is faulty. At this time, the control mainboard 400 controls the second three-way valve of the bronchial passage where the control valve 540 is located to automatically switch to the bronchial passage where the spare second control valve 550 is located, and the second control valve pressure sensor 551 performs pressure detection. If the pressure of the second control valve pressure sensor 551 is normal, the experiment continues normally, otherwise the user is prompted to terminate the experiment in time.

[0069] Corresponding to the number m (n≥1, and is an integer) of chips 800 loaded on the chip transport tray 200, the pressure output module 570 has m gas supply interfaces connected in series on each gas supply passage corresponding to the m chips 800, and the same reaction holes on the m chips 800 are connected to the m gas supply interfaces connected in series on the same gas supply passage. In other words, the pressure output module 570 has n×m gas supply interfaces, and the positions of the gas supply interfaces correspond to the positions of the reaction holes one by one, so that when the chip transport tray 200 is retracted, the gas supply interfaces are connected to the reaction holes on the chips 800 one by one. Taking the illustrated embodiment in which the chip 800 has four reaction holes and four chips are placed side by side as an example, the gas supply component 500 is provided with five branch airways at the rear end of the pressure dividing cylinder 511, one of which (the branch where the second regulating valve 550 is located) is connected to the remaining four branch airways (the branch where the regulating valve 540 is located), and the pressure output module 570 has four gas supply passages, and the pressure output module 570 is provided with four gas supply interfaces on each branch airway.

[0070] In order to ensure the uniformity and consistency of the micro-droplets, it is necessary to monitor whether the parameters of the gas output by the gas supply component 500 meet the set parameters (here, the set parameters can be the parameters input by the user through the display module 300). The gas supply component 500 is provided with a chip pressure sensor 580 for detecting the pressure at the current position on the outlet side of the pressure output module 570. More specifically, a chip pressure sensor 580 is provided corresponding to each gas supply passage of the pressure output module 570 (i.e., the gas supply passage leading to different reaction holes of the chip 800). The chip pressure sensor 580 is used to monitor the pressure level of the bronchial passage connected to the reaction hole of the chip 800 on each gas supply passage in real time. The chip pressure sensor 580 is provided at the position closest to the chip 800 (the outlet side of the pressure output module 570, the front end of the chip 800), so that it can accurately measure and record the slight changes in the pressure of the reaction hole leading to the chip 800, providing key data for the experiment.

[0071] Furthermore, the chip pressure sensor 580 signal is connected to the control mainboard 400. The control mainboard 400 receives the pressure detection data from the chip pressure sensor 580 and controls according to the set parameters, such as the operation of the control valve 540 (or the second control valve 550) and other related components. Each chip pressure sensor 580 corresponds to a gas supply passage and a row of reaction holes. By adjusting the control valve 540 (or the second control valve 550) on the same branch airway by the control mainboard 400, the gas supply pressure of each gas supply passage can be dynamically adjusted in real time according to the pressure value detected and fed back by the chip pressure sensor 580, so as to better respond to the changes in demand during the experiment.

[0072] See also Figure 8 , the air supply assembly 500 of another embodiment of the present application and Figure 7 The difference between the air supply assembly 500 shown in the figure is that the second control valve pressure sensor is omitted on the n+1th bronchial passage where the second control valve 550 is located, and is connected between each control valve 540 and the corresponding control valve pressure sensor 560 through a second three-way valve. In this way, the second control valve 550 can also be used as a spare part for the control valve 540. When the pressure detection data of the control valve sensor 560 on a certain bronchial passage is abnormal, the second three-way valve on the bronchial passage switches the second control valve 550 to work as a spare part, and at this time, the control valve sensor 560 on the bronchial passage performs pressure detection again. If the pressure detection data of the control valve sensor 560 returns to normal, the experiment continues normally, otherwise the user is prompted to terminate the experiment in time.

[0073] In addition to providing gas to the chip 800, the gas supply component 500 can also accurately monitor the experimental pressure in real time and respond to any abnormal situation in a timely manner, thereby improving the reliability and success rate of the experiment. The gas supply component 500 uses the chip pressure sensor 580 to monitor the pressure at different positions in real time, and automatically adjusts the control valve 540 by controlling the main board 400 to maintain the ideal pressure conditions in the experiment. In the gas supply component 500, the chip pressure sensor 580 and the control valve 540 (or the second control valve 550) are used in a one-to-one correspondence, and the real-time feedback mechanism of the data of the chip pressure sensor 580 can be used to prevent and solve problems such as hole blockage.

[0074] Furthermore, the real-time pressure conditions of different positions of the gas supply assembly 500, different bronchial passages, and each reaction hole of the chip 800 are monitored in linkage by the gas generator pressure sensor 530, the control valve pressure sensor 560, and the chip pressure sensor 580. The control mainboard 400 can draw a pressure curve based on this and send it to the display module 300 for display, so that the user can intuitively observe the pressure value and the change in the pressure value. In addition, the control mainboard 400 can set one or more pressure change alarm thresholds, as well as alarm mechanisms corresponding to different pressure change alarm thresholds. When the pressure curve changes significantly and reaches a certain pressure change alarm threshold, the corresponding alarm mechanism can be triggered, for example, the user is prompted on the screen of the display module 300 by different colors, text prompts, and pressure curve jumps and flashes to indicate an abnormal experiment.

[0075] Please refer to Figure 3 , Figure 4 as well as Figures 9 to 11 The real-time image acquisition component 600 includes a camera module 610, an optical lens 620, a connector 630, a light source lamp 640, a light source lamp mounting seat 650 and a light source lamp adjustment driver 660. The camera module 610 is arranged below the chip transport tray 200 along the Z direction, the optical lens 620 is installed on the front end of the lens of the camera module 610 through the connector 630, and the light source lamp 640 is installed above the chip transport tray 200 through the light source lamp mounting seat 650.

[0076] The camera module 610 may use a high frame rate camera to shoot the chip 800 from bottom to top along the Z direction. The high-definition and high-speed image capture capabilities provided by the camera module 610 may capture the rapidly changing experimental conditions in the chip 800.

[0077] The optical lens 620 is disposed above the camera module 610 along the optical axis through the connecting member 630, and is used to focus and guide the light to the photosensitive element of the camera module 610 to ensure the image quality. The optical lens 620 can be, for example, a collimating lens, which collimates the light entering the camera module 610 to improve the imaging effect and image quality.

[0078] The connector 630 connects the optical lens 620 and the camera module 610 to ensure a stable connection of the optical lens 620 and maintain precise optical path alignment.

[0079] The light source lamp 640 is disposed above the chip 800 to provide supplementary light for the camera module 610 disposed below the chip 800 during the shooting process, thereby improving the light brightness of the shooting environment and thus improving the quality of the captured image.

[0080] The light source lamp mounting base 650 is mounted on a driving end of the light source lamp adjusting driving member 660 , so that the light source lamp mounting base 650 is driven by the light source lamp adjusting driving member 660 to move so as to adjust the position of the light source lamp.

[0081] The camera module 610 transmits the captured image data to the control mainboard 400 at high speed via a data line for data processing. The control mainboard 400 can analyze the images captured by the camera module 610 in real time and automatically detect abnormal phenomena in the experiment, such as changes in droplet size, hole blockage, or machine failure. The control mainboard 400 can display the image data in real time on the display module 300, and can also display the analysis results in real time on the display module 300.

[0082] In the illustrated embodiment, a camera module 610 is provided in the real-time image acquisition component 600 for shooting, and one camera module 610 can shoot all the chips 800 on the chip transport tray 200, or the camera module 610 can move in the X direction to shoot all the chips 800 on the chip transport tray 200 one by one in batches. In other embodiments, a plurality of camera modules 610 can be provided in the real-time image acquisition component 600, and the plurality of camera modules 610 are arranged side by side along the X direction, and each camera module 610 is used to shoot one chip 800, so that real-time image acquisition can be performed simultaneously on the plurality of chips 800 on the chip transport tray 200 without adjusting the camera module 610 or moving the position of the camera module 610.

[0083] The real-time image acquisition component 600 displays the experimental process in real time through a high frame rate camera module 610 and an optical lens 620, and transmits the video and images to the display module 300 in real time. The real-time image acquisition component 600 can capture detailed images of each moment to help users accurately evaluate the experimental conditions. The real-time image acquisition component 600 integrates the camera module 610 and the optical lens 620 to capture and display images and videos during the experiment in real time to monitor the generation, size and flow state of droplets. The real-time image acquisition component 600 has a specific optical lens 620 configuration (such as a collimating lens) and a light source lamp 640 design to ensure that clear images can be obtained even in low light conditions.

[0084] The mounting frame 700 includes a mounting base plate 710, a mounting seat 720 and a mounting vertical plate 730. The mounting seat 720 is fixed on the mounting base plate 710 and forms a space between the mounting base plate 710 and the mounting base plate 710 for the chip transport tray 200 to move forward and backward. The mounting vertical plate 730 is fixed on the mounting seat 720 in the vertical direction. The moving mechanism (including the moving guide mechanism) and the power source for the chip transport tray 200 can be fixed on the mounting base plate 710. The gas impurity removal module 510, the gas generator 510, and the regulating valve 540 of the gas supply assembly 500 are all installed on the mounting seat 720. The control main board 400 is fixed in parallel to the mounting vertical plate 730, and the chip pressure sensor 580 is directly arranged on the control main board 400. The mounting plate 730 is provided with an adjustment rail 740 extending in the X direction on the front side, and the light source lamp 640 is movably mounted on the adjustment rail 740 through the light source lamp mounting seat 650, so that the light source lamp 640 can move in the X direction to adjust the position of the light source lamp 640 in the X direction. In the illustrated embodiment, the light source lamp adjustment driving member 660 adopts a rotary motor, and drives the light source lamp 640 to move in the X direction through the cooperation of the light source lamp mounting seat 650 and the adjustment rail 740. In other embodiments, the light source lamp driving member 650 can also adopt a linear motor, which can directly drive the light source lamp mounting seat 650 and the light source lamp 640 to move in the X direction.

[0085] The present application also provides a control method for a single-cell sequencing library construction device, which monitors the pressure anomaly during the experiment through a gas supply component and takes corresponding measures accordingly. Specifically, the control method for a single-cell sequencing library construction device provided by the present application includes the following steps:

[0086] When the pressure detection data of the gas generator pressure sensor 530 at the rear end of the gas generator 510 is abnormal, it is determined that the gas generator 510 is faulty. At this time, the control mainboard 400 controls the first three-way valve to automatically switch to the spare second gas generator 520 (through the pressure-dividing cylinder 511) to communicate with the regulating valve 540, and the gas generator pressure sensor 530 performs pressure detection again. If the pressure detection data of the gas generator pressure sensor 530 returns to normal, the experiment continues normally, otherwise the user is prompted to terminate the experiment in time.

[0087] When the pressure detection data of the gas generator pressure sensor 530 at the rear end of the gas generator 510 is normal, but the pressure detection data of the control valve pressure sensor 560 at the rear end of a certain control valve 540 is abnormal, the control main board 400 controls the second three-way valve on the bronchial passage where the control valve 540 is located to automatically switch to the spare second control valve 550 connected to the pressure output module 570, and the second control valve pressure sensor 551 performs pressure detection (such as Figure 7 ) or the corresponding control valve pressure sensor 560 performs pressure detection again (such as Figure 8 If the pressure detection data returns to normal, the experiment continues normally, otherwise the user is prompted to terminate the experiment in time.

[0088] When the pressure detection data of the gas generator pressure sensor 530 at the rear end of the gas generator 510 and all the control valve pressure sensors 560 (and the second control valve pressure sensor 551) at the rear end of the control valve 540 are normal, but the pressure detection data of the chip pressure sensor 580 at the rear end of the pressure output module 570 is abnormal, it is often due to an abnormality in the gas path between the rear end of the control valve pressure sensor 560 and the chip 800. This fault is a fault that cannot be self-eliminated, and the user needs to be prompted to terminate the experiment immediately and check the relevant gas path components.

[0089] In view of the problem in the prior art that instrument failures or changes in experimental conditions are often not discovered until after the experiment is over, resulting in a large number of experimental repetitions and sample waste, the gas supply assembly of the present application can monitor pressure changes during the experiment in real time and promptly detect anomalies such as hole blockage or unstable air pressure, thereby allowing the user to adjust or terminate the experiment immediately. This instant feedback mechanism significantly improves the reliability and success rate of the experiment.

[0090] The pneumatic system of the prior art has the following problems: the power to drive the liquid flow of the chip usually relies on the pneumatic system, but the pressure detection setting of the pneumatic system of the prior art is unreasonable, and some abnormalities during the experiment often fail to trigger the pressure sensor or the self-test device to alarm, resulting in the failure of the experiment. For example, the pressure sensor is installed at the rear end of the control valve. When the failure of the air supply component occurs at the rear end of the pressure sensor to the chip input port, it cannot be sensed, so the pressure display is normal even when the failure occurs, resulting in the experiment continuing despite failure, wasting specimens and reagents.

[0091] The gas supply component of the present application is used to provide gas to the chip to form droplets. The gas supply component includes a gas generator, a control valve and a pressure output module in sequence along the gas flow direction. The pressure output module supplies gas to the reaction holes of the chip, and a chip pressure sensor for detecting the airway pressure is arranged on the outlet side of the pressure output module. The chip pressure sensor can transmit feedback data to the control mainboard, and the control mainboard adjusts the control valve output through the PID algorithm to maintain the pressure within the set range, thereby achieving high-precision pressure control.

[0092] The pneumatic system of the prior art also has the following problems: the gas supply component cannot self-check in real time, and when a fault occurs during the experiment, the specific fault point cannot be accurately located in real time. In the gas supply component and single-cell sequencing library construction equipment of the present application, a multi-pressure sensor linkage solution is adopted, and a high-precision pressure sensor is installed in each fault section, and pressure sensors are arranged at the rear end of the gas generator, the rear end of each control valve, and the rear end of each airway of the pressure output module. The comprehensive linkage of multiple pressure sensors effectively solves the problem that certain abnormalities cannot trigger the alarm of the pressure sensor or the self-checking device and cannot accurately locate the specific fault point in real time, thereby realizing the precise real-time detection of pressure and the rapid positioning of the fault location.

[0093] The prior art gas supply assembly does not have a self-troubleshooting function, and a failure usually means that the experiment has failed. The single-cell sequencing library construction device and its control method of the present application, by setting a second gas generator, a second regulating valve and corresponding control logic, can provide corresponding automatic troubleshooting solutions for different faults. In addition, in the single-cell sequencing library construction device of the present application, the real-time image acquisition component integrates high frame rate imaging and precise optical monitoring, which greatly enhances the monitoring efficiency and quality control capabilities during the experiment. Through real-time visual feedback, users can adjust experimental parameters in time or terminate the experiment when necessary, thereby effectively avoiding the loss of precious samples and improving the accuracy of the data. The real-time images and videos provided by the real-time image acquisition component intuitively show the key dynamics of the experimental process, such as the generation of droplets and the flow of microchannels. This enables the experimenter to intuitively evaluate the experimental status and quickly identify problems, such as droplet size discrepancies or air leaks, further enhancing the control accuracy of the experiment.

[0094] The single-cell sequencing library building equipment of the present application, through the comprehensive application of pressure monitoring and visual monitoring systems, can reduce experimental failures caused by equipment failures or improper experimental conditions, ensure real-time quality control of the experiment during operation, and enable users to choose to terminate the experiment at any time when an experimental abnormality is found, which will significantly improve the operational efficiency and data quality of droplet single-cell sequencing and ensure the accuracy of experimental results. Repeated experiments and sample waste are common and expensive problems in the prior art. The single-cell sequencing library building equipment of the present application can promptly discover and solve problems, avoiding repeated experiments and sample waste. Accurate monitoring also means that variations in the data collection process are reduced, improving the repeatability and reliability of the data. This is particularly important for key applications such as gene expression analysis, because the accuracy of the data directly affects the interpretation and subsequent application of research results.

[0095] The gas supply assembly and single-cell sequencing library construction equipment of this application improve the existing single-cell sequencing technology and achieve significant technical effects in terms of real-time quality control and experimental efficiency, which are specifically reflected in the following aspects:

[0096] (1) Improved operational flexibility: Allowing users to adjust experimental settings based on real-time feedback provides greater operational flexibility. This is particularly valuable for processing complex or variable biological samples because it allows users to optimize experimental conditions to obtain the best results.

[0097] (2) Save costs and resources: Real-time monitoring reduces repeated experiments and waste of resources due to experimental failures, especially when using precious or difficult-to-obtain samples. In addition, improving the success rate of experiments can speed up research progress and shorten project timelines.

[0098] (3) Expanding application areas: By improving quality control and data reliability, single-cell technology can be used in clinical research and precision medicine. For example, it can be used for single-cell analysis in the process of disease diagnosis, where the accuracy and reliability of data are crucial.

[0099] In summary, the single-cell sequencing library construction equipment of the present application adopts the innovative integration of real-time pressure monitoring and visual monitoring systems, which not only significantly improves the operational flexibility and experimental success rate of single-cell sequencing, but also effectively reduces costs and improves efficiency by reducing repeated experiments and improving data quality, bringing new possibilities for the application and development of single-cell sequencing technology. The gas supply component and single-cell sequencing library construction equipment of the present application solve a long-standing problem in droplet single-cell sequencing, namely how to achieve real-time quality control; by introducing an advanced monitoring system, it is possible to detect and adjust experimental conditions in real time throughout the entire experiment, significantly reducing sample loss and improving data quality. This innovation not only improves the efficiency and reliability of single-cell sequencing, but also provides a powerful tool for future biomedical research.

[0100] 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.

[0101] 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 gas supply assembly for supplying gas to a chip (800) to form droplets, characterized in that: The gas supply assembly (500) includes a gas generator (510), a gas generator pressure sensor (530), a control valve (540) and a pressure output module (570) in sequence along the gas flow direction; the pressure output module (570) supplies gas to the reaction hole of the chip (800); and a chip pressure sensor (580) for detecting airway pressure is arranged on the outlet side of the pressure output module (570); Corresponding to the number n of reaction holes on each chip (800), n air ducts are provided from the gas generator (510) to the pressure output module (570), one control valve (540) is provided on each of the n air ducts, n air supply passages are provided in the pressure output module (570) corresponding to the n air ducts, and one chip pressure sensor (580) is provided on the outlet side of each air supply passage, wherein n is an integer greater than or equal to 1; the control valve (540) adjusts the air supply pressure of the air duct according to the pressure detection data of the chip pressure sensor (580) on the same air duct; The gas supply assembly (500) is also provided with a second gas generator (520), and the gas generator (510) and the second gas generator (520) are connected to the gas path leading to the control valve (540) via a first three-way valve, so that the gas generator (510) and the second gas generator (520) can be connected to the control valve (540) in an alternative manner.

2. The air supply assembly according to claim 1, characterized in that: In the pressure output module (570), m gas supply interfaces are arranged in series on each gas supply passage corresponding to the m chips (800), and the same reaction holes on the m chips (800) are connected to the m gas supply interfaces connected in series on the same gas supply passage, wherein m is an integer greater than or equal to 1.

3. The air supply assembly according to claim 1, characterized in that: The gas supply assembly (500) is also provided with a control valve pressure sensor (560), and the control valve pressure sensor (560) is connected to the gas path from each control valve (540) to the pressure output module (570).

4. The air supply assembly according to claim 3, characterized in that: An n+1th airway is also provided between the gas generator (510) and the pressure output module (570), and a second regulating valve (550) is provided on the n+1th airway. The second regulating valve (550) is respectively connected to the airway where each regulating valve (540) is located through a second three-way valve, so that the airway where any regulating valve (540) is located can be switched to the airway where the second regulating valve (550) is located through the corresponding second three-way valve and connected to the pressure output module (570).

5. A single-cell sequencing library construction device, characterized in that: The invention comprises a housing (100), a chip transport tray (200), a display module (300), a control main board (400) and an air supply assembly (500) according to any one of claims 1 to 4, wherein the chip transport tray (200) is used to carry a chip (800) and is arranged in the housing (100) so as to be movable outward and inward, the display module (300) is arranged on the housing (100), and the control main board (400) and the air supply assembly (500) are arranged in the housing (100); the display module (300), the control valve (540) and the chip pressure sensor (580) are connected to the control main board (400) by signals, and the control main board (400) receives pressure detection data from the chip pressure sensor (580) and controls the control valve (540) to adjust the air supply pressure.

6. The single-cell sequencing library construction device according to claim 5, characterized in that: The control mainboard (400) sends the pressure detection data of the chip pressure sensor (580) to the display module (300) for display; the control mainboard (400) sets a pressure change alarm threshold value so that when the pressure detection data of the chip pressure sensor (580) reaches the pressure change alarm threshold value, an abnormality is prompted on the display module (300).

7. The single-cell sequencing library construction device according to claim 5, characterized in that: The single-cell sequencing library building device also includes a real-time image acquisition component (600) disposed in the housing (100), and the real-time image acquisition component (600) includes a camera module (610). The camera module (610) is disposed below the chip transport tray (200) to photograph the chip (800) on the chip transport tray (200) from bottom to top.

8. A method for controlling the single-cell sequencing library construction device according to claim 5, comprising the following steps: When the pressure detection data of the chip pressure sensor (580) is abnormal, the user is prompted to terminate the experiment.

9. The control method of the single-cell sequencing library construction device according to claim 8, characterized in that: The following steps are also included: When the pressure detection data of the gas generator pressure sensor (530) is abnormal, the control main board (400) controls the first three-way valve to switch to the second gas generator (520) to communicate with the control valve (540), and the gas generator pressure sensor (530) performs pressure detection again; If the pressure detection data of the gas generator pressure sensor (530) returns to normal, the experiment continues normally; otherwise, the user is prompted to terminate the experiment.

10. The control method of the single-cell sequencing library construction device according to claim 9, characterized in that: The air supply assembly (500) is further provided with a second regulating valve (550), a regulating valve pressure sensor (560) and a second regulating valve pressure sensor (551); the regulating valve pressure sensor (560) is connected to the bronchial passage from each regulating valve (540) to the pressure output module (570); the second regulating valve (550) and the second regulating valve pressure sensor (551) are connected to the bronchial passage from each regulating valve (540) to the pressure output module (570) via a second three-way valve; The control method of the single-cell sequencing library building device also includes the following steps: When the pressure detection data of the gas generator pressure sensor (530) is normal and the pressure detection data of a certain control valve pressure sensor (560) is abnormal, the control main board (400) controls the second three-way valve on the bronchial passage where the control valve pressure sensor (560) is located to switch to the bronchial passage where the second control valve (550) is located to be connected to the pressure output module (570), and the second control valve pressure sensor (551) performs pressure detection; If the pressure detection data of the second control valve pressure sensor (551) is normal, the experiment continues normally; otherwise, the user is prompted to terminate the experiment.

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