Gas supply assembly and single cell sequencing library building instrument

Abstract

The utility model provides a gas supply assembly and a single cell sequencing library building instrument, the gas supply assembly is used for providing gas for a chip to form micro-droplet liquid, the gas supply assembly sequentially comprises a gas generator, a regulation and control valve and a pressure output module along the gas flowing direction, the pressure output module supplies gas to a reaction hole of the chip, and the gas generator is used for generating the micro-droplet liquid. The outlet side of the pressure output module is provided with a chip pressure sensor used for detecting the pressure of an airway, and the regulation and control valve adjusts the pressure of gas leading to the pressure output module according to pressure detection data of the chip pressure sensor. According to the gas supply assembly and the single cell sequencing library building instrument, the chip pressure sensor is used for monitoring the pressure change in the experiment process in real time and finding abnormity in time, and feedback data of the chip pressure sensor is used for controlling and adjusting the output of the regulation and control valve, so that high-precision pressure control can be realized; a user can adjust or stop the experiment in real time according to the abnormity of pressure detection, and the reliability and success rate of the experiment are remarkably improved.

Description

Technical Field

[0001] This application relates to the technical field of sequencers, and more particularly to a gas supply component and a single-cell sequencing library construction instrument. Background Art

[0002] Currently, single-cell sequencing technologies are 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 by co-encapsulating single cells and reaction reagents in tiny oil droplets. These droplets independently serve 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 plate-based method. Each droplet usually contains a unique DNA molecule with a barcode for identifying RNA or DNA molecules derived from the same cell in subsequent genomic sequencing. The main advantages of this technology are its high throughput and low sample input requirements.

[0003] However, the droplet method has obvious limitations in actual operation. In the prior art, the pressure sensor for detecting faults in the gas supply component is installed at the rear end of the regulating valve. When a fault in the gas supply component occurs between the rear end of this pressure sensor and the chip input port, the pressure sensor cannot sense the fault. Therefore, there will be a situation where a fault occurs but the pressure display is normal, resulting in the experiment continuing despite the failure and wasting specimens and reagents. Summary of the Invention

[0004] To solve the existing technical problems, this application provides a gas supply component and a single-cell sequencing library construction instrument, which can avoid the situation of neglecting to prompt when a fault occurs and provide more precise pressure control.

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

[0006] On the one hand, the embodiment of this application provides a gas supply component for supplying gas to a chip to form microdroplets; the gas supply component sequentially includes a gas generator, a regulating valve, and a pressure output module 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, and the regulating valve adjusts the gas pressure leading to the pressure output module according to the pressure detection data of the chip pressure sensor.

[0007] In one embodiment, the gas supply assembly further includes a second gas generator and a gas generator pressure sensor. 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, and the gas generator pressure sensor is connected to the gas path between the first three-way valve and the control valve.

[0008] In one embodiment, there are multiple branch airways leading from the gas generator to the pressure output module. A control valve is provided on each branch airway, and a corresponding number of gas supply passages are provided in the pressure output module corresponding to the branch airways. A chip pressure sensor is provided on the outlet side of each gas supply passage; a plurality of gas supply interfaces are connected in series on each gas supply passage of the pressure output module, and each gas supply interface communicates with a reaction hole of the chip.

[0009] In one embodiment, a chip pressure sensor is provided on the outlet side of each gas supply passage of the pressure output module, and each control valve adjusts the gas pressure of the gas supply passage leading to the pressure output module according to the pressure detection data of the chip pressure sensor on the corresponding branch airway.

[0010] In one embodiment, the gas supply assembly further includes a second control valve and a control valve pressure sensor. The control valve pressure sensor is connected to the branch airway between each control valve and the pressure output module, and the second control valve is bypassed to the branch airway where each control valve is located through a second three-way valve.

[0011] In one embodiment, the control valve and the second control valve are proportional valves.

[0012] In one embodiment, the gas supply assembly further includes a gas generator pressure sensor, and the gas generator pressure sensor is connected to the gas path between the gas generator and the control valve;

[0013] And / or, the gas supply assembly further includes a control valve pressure sensor, and the control valve pressure sensor is connected to the gas path between the control valve and the pressure output module.

[0014] In one embodiment, the gas supply assembly further includes 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;

[0015] And / or, the gas supply assembly further includes a second control valve, and the second control valve is connected to the gas path of the control valve leading to the pressure output module through a second three-way valve.

[0016] On the other hand, an embodiment of the present application provides a single-cell sequencing library construction instrument, including a shell, a chip transport tray, 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 control main board and the gas supply assembly are arranged in the shell; the control valve and the chip pressure sensor signals are connected to the control main board.

[0017] In one embodiment, the single-cell sequencing library construction instrument further includes a display module, which is disposed on the housing and has a signal connected to the control main board.

[0018] The gas supply assembly, single-cell sequencing library construction instrument 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 instrument of the present application, the pressure changes during the experiment are monitored in real time by the chip pressure sensor arranged on the outlet side of the pressure output module, so that the pressure and pressure changes can be monitored in real time at the position closest to the chip, and the feedback data of the chip pressure sensor controls the output of the regulating valve to maintain the pressure within the set range, thereby achieving high-precision pressure control; and, real-time monitoring of the pressure and pressure changes at the position closest to the chip can promptly detect abnormalities such as hole blockage or unstable air pressure, thereby allowing the user to immediately adjust or terminate the experiment, and avoid as much as possible the occurrence of failures without prompts. This instant feedback mechanism significantly improves the reliability and success rate of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the structure of a single-cell sequencing library construction instrument in a first state according to an embodiment of the present application;

[0020] Figure 2 for Figure 1 A schematic diagram of the structure of the single-cell sequencing library construction instrument in the second state;

[0021] Figure 3 for Figure 1 Schematic diagram of the internal structure of the single-cell sequencing library construction instrument;

[0022] Figure 4 for Figure 3 A schematic diagram of the internal structure of the single-cell sequencing library construction instrument from another angle;

[0023] Figure 5 This is a schematic structural diagram of an air supply assembly according to an embodiment of the present application;

[0024] Figure 6 for Figure 5 A schematic structural diagram of the air supply assembly from another angle;

[0025] Figure 7Schematic structural framework diagram of the connection between the gas supply component and the chip according to an embodiment of the present application;

[0026] Figure 8 Schematic structural framework diagram of the connection between the gas supply component and the chip according to another embodiment of the present application;

[0027] Figure 9 is Figure 3 Schematic structural diagram of the real-time image acquisition component in

[0028] Figure 10 is Figure 9 Schematic structural diagram of the real-time image acquisition component from another angle in

[0029] Figure 11 is Figure 9 Schematic bottom view structural diagram of the real-time image acquisition component in

[0030] The reference numerals of each component in the figure are as follows:

[0031] Housing 100;

[0032] Chip transport tray 200;

[0033] Display module 300;

[0034] Control main board 400;

[0035] Gas supply component 500 (wherein, gas generator 510, second gas generator 520, pressure dividing cylinder 511, gas generator pressure sensor 530; regulating valve 540, second regulating valve 550, regulating valve pressure sensor 560, second regulating valve pressure sensor 551; pressure output module 570; chip pressure sensor 580); Real-time image acquisition component 600 (wherein, camera module 610, optical lens 620, connecting member 630, fill light 640, fill light mounting base 650, fill light adjustment driving member 660);

[0036] Mounting frame 700 (wherein, mounting base plate 710, mounting seat 720, mounting vertical plate 730, adjustment slide rail 740);

[0037] Chip 800. Detailed implementation manners

[0038] The technical solution of the present application will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments.

[0039] 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 terms used in the specification of this application are for the purpose of describing specific embodiments only 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 of the related listed items.

[0040] In the description of this application, it should be understood that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to this application. In the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0041] 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 directly connected, or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0042] Microfluidics refers to a technology for manipulating fluids in a micron-scale space. This technology can miniaturize the basic functions of chemical, biological, and other laboratories onto a chip of a few square centimeters, and is therefore also known as Lab-on-a-chip. The microfluidic chip system can manipulate the flow of fluids in tiny channels or components with dimensions ranging from dozens to hundreds of microns, and the volume of the fluid 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 onto a very small chip. A network of microchannels formed by controllable fluids runs through the microfluidic system, realizing the various functions of a conventional biochemical laboratory while reducing the cost of analysis and detection, accelerating the reaction speed, improving the reaction efficiency, and making the experiment more controllable.

[0043] Generating droplets on a microfluidic chip is a process in which one-phase fluid is dispersed in another immiscible or partially immiscible fluid. For two immiscible liquids, one is used as the continuous phase and the other as the dispersed phase, and the dispersed phase is in a tiny volume (10 -15 ~10 -9In the form of unit (L), it is dispersed in the continuous phase to form droplets.

[0044] During the droplet generation process, there may sometimes be foreign objects in a single microchannel. These foreign objects may cause blockage, or although they do not cause blockage, they will affect the flow rate. The presence of foreign objects will affect the flow rate of the solution in the single microchannel, easily resulting in uneven droplet sizes, or discontinuous droplet intervals due to too slow flow rate. Sometimes, there will also be an unstable pressure phenomenon in the single microchannel. The unstable pressure will affect the solution flow rate, making the generated droplet sizes uneven and the intervals discontinuous. The prior art cannot monitor and control these abnormal phenomena in real time, cannot perform real-time quality control on the generated droplet quality, resulting in abnormal droplets mixing with normal droplets after entering the droplet storage cavity. If the number of abnormal droplets is large, they can be discovered through size differences under a microscope, but even if discovered, there is no way to remedy them, and only a new experiment can be carried out, causing waste of specimens, especially precious trace samples; at the same time, the reagent consumables for single-cell sequencing are also wasted, resulting in waste of costs and time. 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 with the next step of amplification and library construction, resulting in incorrect results, seriously affecting scientific research and clinical diagnosis and treatment. In both of the above cases, it is only possible to observe and discover under a microscope after the experiment is over and the machine is turned off. The prior art cannot perform real-time quality control on the droplet quality before turning off the machine, let alone perform quality intervention on abnormal droplets during the experiment.

[0045] The single-cell sequencing library construction instrument of the present application applies droplet microfluidic technology to complete the capture, separation, and labeling of single cells in one body, and can be used in conjunction with a single-cell sequencing kit to achieve flexible, stable, and efficient single-cell sequencing library preparation and other work.

[0046] Please refer to Figure 1 and Figure 2 , the single-cell sequencing library construction instrument of an embodiment of the present application includes a housing 100, a chip transport tray 200, a display module 300, a control main board 400, a gas supply component 500, and a real-time image acquisition component 600 (please refer to Figures 3 to 8 in combination). Among them, the chip transport tray 200 is used to carry the chip 800, and is movably arranged in the housing 100 through a moving mechanism, and can be moved outwards and inwards 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 to the user; the control main board 400, the gas supply component 500, and the real-time image acquisition component 600 are all fixedly installed in the housing 100 through a mounting bracket 700. The single-cell sequencing library construction instrument 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.

[0047] The housing 100 can be formed by enclosing multiple plates to create a space for accommodating components such as the control main board 400, the gas supply assembly 500, and the real-time image acquisition assembly 600. In the illustrated embodiment, the front end of the housing 100 has an upwardly inclined opening, so that the screen of the display module 300 can be installed at the opening in a tilted upward manner, and the tilt angle of the screen conforms to the viewing habits of users, facilitating viewing and / or operation. The display module 300 can use a touch screen, or there are operation buttons beside the screen, so that users can input instructions by touching the screen or operating the buttons. That is, the display module 300 can have both a display function and an input function.

[0048] The single-cell sequencing library preparation instrument has two states, as shown respectively in Figure 1 and Figure 2 . In Figure 1 the first state shown, the chip transport tray 200 retracts into the housing 100, and the single-cell sequencing library preparation instrument can work; in Figure 2 the second state shown, the chip transport tray 200 extends forward in the Y direction outside the housing 100 to place a chip 800 on or remove a chip 800 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 arranged below the display module 300, and 4 chips 800 can be placed side by side thereon in the X direction; each chip 800 has 4 reaction holes.

[0049] The control main board 400 is the centralized control core of the entire single-cell sequencing library preparation instrument, responsible for coordinating and managing the functions of each component, and through the control of the control main board 400, each part works together to complete single-cell sequencing library preparation. For example, the display module 300, the gas supply assembly 500, and the real-time image acquisition assembly 600 are all signal-connected and controlled by the control main board 400.

[0050] The single-cell sequencing library preparation instrument uses air flow to drive the movement of fluids, so that the fluids form micro-droplets. The gas supply assembly 500 is used to supply gas to the chips 800 on the chip transport tray 200 to form micro-droplets.

[0051] Please refer to Figures 3 to 7, in an embodiment of the present application, the gas supply assembly 500 sequentially includes a gas generator 510, a regulating valve 540, and a pressure output module 570 along the gas flow direction. After the gas sequentially passes through the gas generator 510 and the regulating valve 540, the pressure output module 570 supplies gas to the reaction holes of the chip 800. Both the gas generator 510 and the regulating valve 540 are signal-connected to the control main board 400 and controlled by the control main board 400. The pressure output module 570 is correspondingly arranged above the chip transport tray 200 in the Z direction. When the chip transport tray 200 retracts into the housing 100 in the Y direction, the gas supply interface at the lower end of the pressure output module 570 is vertically connected to the reaction holes of the chip 800 in the Z direction.

[0052] To further improve the gas supply quality of the gas supply assembly, a gas purification module (not shown in the figure) can be provided at the front end of the gas generator 510. The gas purification module is used to remove moisture or other impurities in the gas. Removing moisture in 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.

[0053] The gas generator 510 provides a stable and adjustable air pressure for use in the entire single-cell sequencing library construction experiment process. The gas generator 510 is a key device for maintaining the required pressure environment for the experiment and is the basis for ensuring that the experimental conditions meet the preset standards.

[0054] At the same time, a second gas generator 520 is also provided in the gas supply assembly 500. The gas generator 510 and the second gas generator 520 are connected to the front end of the gas path leading to the regulating valve 540 through a first three-way valve (not labeled). The second gas generator 520 is signal-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 regulating valve 540 in an alternative manner. That is, the second gas generator 520 can be used as a spare part for the gas generator 510. When it is determined that the gas generator 510 is abnormal, the gas supply assembly 500 switches from supplying gas to the regulating valve 540 by the gas generator 510 to supplying gas to the regulating valve 540 by the second gas generator 520.

[0055] Corresponding to each chip 800, there are multiple reaction holes. The gas sources generated by the gas generator 510 and the second gas generator 520 are divided from one main air duct into multiple branch air ducts by the pressure dividing cylinder 511 and then lead to the regulating valve 540.

[0056] To more precisely 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., on the gas path where the first three-way valve leads to the regulating 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 signal of the gas generator pressure sensor 530 is connected to the control main board 400 to send the pressure detection data to the control main board 400.

[0057] More specifically, in the initial state, the first three-way valve connects the gas generator 510 to 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 main board 400 controls the first three-way valve to automatically switch to the standby 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 a timely manner. The regulating valve 540 is used to control the rate and amount of gas flowing into each reaction well of the chip 800 to ensure the uniformity and stability of the experimental conditions. The regulating valve 540 can be a proportional pressure valve, for example.

[0058] Corresponding to the number n (n≥1 and is an integer) of reaction wells on each chip 800, the main air duct is divided into n + 1 branch air ducts by the pressure dividing cylinder 511 from the gas generator 510 leading to the chip 800. Among them: on each of the n branch air ducts, a regulating valve 540 and a regulating valve pressure sensor 560 are provided and connected to the pressure output module 570; on the (n + 1)th branch air duct, a second regulating valve 550 and a second regulating valve pressure sensor 551 are provided and bypassed to the remaining n branch air ducts. The pressure output module 570 is correspondingly provided with n gas supply paths, and a chip pressure sensor 580 is provided on the outlet side of each gas supply path (branch air duct).

[0059] On the (n + 1)-th airway branch, the second regulating valve 550 and the second regulating valve pressure sensor 551 are respectively connected to the remaining n airway branches through n second three-way valves (not labeled) to the front end of the pressure output module 570. The second regulating valve 550 is signal-connected to the control main board 400, and the control main board 400 controls one of the regulating valves 540 and the second regulating valve 550 to regulate the flow rate and flow volume of the gas in the gas path leading to the pressure output module 570 in an alternative manner. That is to say, the second regulating valve 550 can be used as a spare part for the regulating valve 540. When it is determined that a certain regulating valve 540 is abnormal, the gas supply component 500 switches the gas source provided by the regulating valve 540 to the corresponding gas supply path of the pressure output module 570 to the gas source provided by the second regulating valve 550 to the corresponding gas supply path of the pressure output module 570.

[0060] To more accurately determine whether the regulating valve 540 is abnormal, a regulating valve pressure sensor 560 is connected to the rear end of each regulating valve 540. The regulating valve pressure sensor 560 is used to monitor the pressure at the current position in real time, and the regulating valve pressure sensor 560 is signal-connected to the control main board 400 to send the pressure detection data to the control main board 400.

[0061] More specifically, when the regulating valve pressure sensor 560 at the rear end of a certain regulating valve 540 detects abnormal pressure data, it is determined that the regulating valve 540 fails. At this time, the control main board 400 controls the second three-way valve of the airway branch where the regulating valve 540 is located to automatically switch to the airway branch where the spare second regulating valve 550 is located, and the second regulating valve pressure sensor 551 performs pressure detection. If the pressure of the second regulating valve pressure sensor 551 is normal, the experiment continues normally; otherwise, the user is prompted to terminate the experiment in time.

[0062] Corresponding to the number m of chips 800 loaded on the chip transport tray 200 (n ≥ 1 and is an integer), m gas supply interfaces are serially arranged on each gas supply path in the pressure output module 570 corresponding to the m chips 800. The same reaction holes on the m chips 800 communicate with the m gas supply interfaces serially arranged on the same gas supply path. In other words, there are n × m gas supply interfaces on the pressure output module 570, and the positions of the gas supply interfaces correspond one-to-one with the positions of the reaction holes, so that in the retracted state of the chip transport tray 200, the gas supply interfaces are connected to the reaction holes on the chips 800 one-to-one. Taking the example where the chip 800 has 4 reaction holes and 4 chips are placed side by side in the illustrated embodiment, the gas supply component 500 sets 5 airway branches at the rear end of the pressure dividing cylinder 511, where 1 airway branch (the airway branch where the second regulating valve 550 is located) is bypassed to the remaining 4 airway branches (the airway branches where the regulating valves 540 are located), the pressure output module 570 has 4 gas supply paths, and the pressure output module 570 sets 4 gas supply interfaces on each airway branch.

[0063] To ensure the uniformity and consistency of 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, for 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), a chip pressure sensor 580 is provided. The chip pressure sensor 580 is used to monitor in real time the pressure level of the branch airway connected to the reaction hole of the chip 800 on each gas supply passage. The chip pressure sensor 580 is arranged 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 as to accurately measure and record the minute pressure changes leading to the reaction holes of the chip 800, providing key data for the experiment.

[0064] Furthermore, the chip pressure sensor 580 is signal-connected to the control main board 400. The control main board 400 receives the pressure detection data from the chip pressure sensor 580 and performs control according to the set parameters, such as operating the regulating valve 540 (or the second regulating 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 regulating valve 540 (or the second regulating valve 550) on the same branch airway through the control main board 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 demand changes during the experiment.

[0065] Please refer to Figure 8 , the difference between the gas supply component 500 of another embodiment of the present application and the gas supply component 500 shown in Figure 7 is that the second regulating valve pressure sensor is omitted on the (n + 1)-th branch airway where the second regulating valve 550 is located, and it is connected to between each regulating valve 540 and the corresponding regulating valve pressure sensor 560 through a second three-way valve. In this way, the second regulating valve 550 can also be used as a spare part for the regulating valve 540. When the pressure detection data of the regulating valve sensor 560 on a certain branch airway is abnormal, the second three-way valve on this branch airway switches the second regulating valve 550 to work as a spare part. At this time, the pressure is still detected again by the regulating valve sensor 560 on this branch airway. If the pressure detection data of the regulating valve sensor 560 returns to normal, the experiment continues normally; otherwise, the user is prompted to terminate the experiment in time.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] The connecting member 630 connects the optical lens 620 and the camera module 610, ensuring a stable connection of the optical lens 620 and maintaining an accurate optical path alignment.

[0072] The light source lamp 640 is arranged above the chip 800 to supplement light for the shooting process of the camera module 610 arranged below the chip 800, improving the illumination brightness of the shooting environment, thereby enhancing the quality of the captured images.

[0073] The light source lamp mounting seat 650 is mounted on the driving end of the light source lamp adjusting driving member 660, so that the light source lamp adjusting driving member 660 drives the light source lamp mounting seat 650 to move to adjust the position of the light source lamp.

[0074] The camera module 610 transmits the captured image data to the control main board 400 at high speed through a data cable for data processing. The control main board 400 can perform real-time analysis on the images captured by the camera module 610, automatically detecting abnormal phenomena in the experiment, such as changes in droplet size, blocked holes, or machine failures, etc. The control main board 400 can display the image data on the display module 300 in real time, and can also display the analysis results on the display module 300 in real time.

[0075] In the illustrated embodiment, one camera module 610 is provided in the real-time image acquisition component 600 for shooting. 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 and shoot all the chips 800 on the chip transport tray 200 one by one in batches. In other embodiments, multiple camera modules 610 can be provided in the real-time image acquisition component 600. The multiple camera modules 610 are arranged side by side in the X direction, and each camera module 610 is used to shoot one chip 800, so that real-time image acquisition of multiple chips 800 on the chip transport tray 200 can be performed simultaneously without adjusting the position of the camera module 610 or moving the camera module 610.

[0076] The real-time image acquisition component 600 displays the experimental process in real time through the high-frame-rate camera module 610 and the 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, helping the user accurately evaluate the experimental conditions. The real-time image acquisition component 600 integrates the camera module 610 and the optical lens 620, capturing and displaying the images and videos in the experimental process in real time to monitor the generation, size, and flow state of the droplets. The specific optical lens 620 configuration (such as a collimating lens) and the design of the light source lamp 640 of the real-time image acquisition component 600 are used to ensure clear images can be obtained even under low-light conditions.

[0077] The mounting bracket 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 seat 720 and the mounting base plate 710 for the chip transport tray 200 to move back and forth. The mounting vertical plate 730 is fixed on the mounting seat 720 in the vertical direction. The moving mechanism (including the moving guiding mechanism) and the power source provided for the chip transport tray 200 can both be fixed on the mounting base plate 710. The gas purification module 510, the gas generator 510, and the regulating valve 540 of the gas supply assembly 500 are all mounted on the mounting seat 720. The control main board 400 is fixedly parallel to the mounting vertical plate 730, and the chip pressure sensor 580 is directly arranged on the control main board 400. The mounting vertical plate 730 is provided with an adjustment slide rail 740 extending in the X direction on the front side. The light source lamp 640 is movably mounted on the adjustment slide 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 slide 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.

[0078] Aiming at the problem that instrument failures or changes in experimental conditions in the prior art are often discovered only after the experiment is completed, resulting in a large number of repeated experiments and sample waste, the gas supply assembly of the present application can monitor the pressure change in the experimental process in real time and detect abnormalities in time, such as blocked holes or unstable air pressure, thereby allowing the user to adjust or abort the experiment immediately. This immediate feedback mechanism significantly improves the reliability and success rate of the experiment.

[0079] The prior art pneumatic system has the following problems: The power for driving the liquid flow of the chip usually depends on the pneumatic system, but the pressure detection setting of the prior art pneumatic system is unreasonable, and some abnormalities during the experiment often cannot trigger the alarm of the pressure sensor or the self-check device, resulting in experimental failure. For example, the pressure sensor is installed at the rear end of the regulating valve. When a failure of the gas supply assembly occurs between the rear end of this pressure sensor and the chip input port, it cannot be sensed. Therefore, there will be a situation where the pressure shows normal while a failure occurs, resulting in the continuation of the experiment despite the failure, wasting specimens and reagents.

[0080] The gas supply component of the present application is used to supply gas to a chip to form microdroplets. The gas supply component sequentially includes a gas generator, a regulating valve, and a pressure output module 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 main board, and the control main board adjusts the output of the regulating valve through a PID algorithm to maintain the pressure within a set range, achieving high-precision pressure control.

[0081] The existing air pressure system still has the following problems: The gas supply component cannot perform real-time self-check. When a failure occurs during the experiment, the specific failure point cannot be accurately located in real time. In the gas supply component and the single-cell sequencing library construction instrument of the present application, a multi-pressure sensor linkage scheme is adopted. High-precision pressure sensors are arranged at each failure section, and pressure sensors are arranged at positions such as the rear end of the gas generator, the rear end of each regulating valve, and the rear end of each airway of the pressure output module. The comprehensive linkage of multiple pressure sensors effectively solves the problems that some abnormalities cannot trigger the alarm of the pressure sensor or the self-check device and the specific failure point cannot be accurately located in real time, thereby achieving accurate real-time pressure detection and rapid positioning of the failure occurrence location.

[0082] The gas supply component of the prior art does not have a self-fault exclusion function, and a failure usually means the failure of the experiment. In the single-cell sequencing library construction instrument and its control method of the present application, by setting a second gas generator, a second regulating valve, and corresponding control logics, corresponding automatic fault exclusion solutions can be provided for different faults. In addition, in the single-cell sequencing library construction instrument of the present application, the real-time image acquisition component integrates high-frame-rate imaging and precise optical monitoring, greatly enhancing the monitoring efficiency and quality control ability during the experiment. Through real-time visual feedback, the user can timely adjust the experimental parameters or terminate the experiment when necessary, thereby effectively avoiding the loss of precious samples and improving the accuracy of data. The real-time images and videos provided by the real-time image acquisition component intuitively show the key dynamics during the experiment, such as the generation of droplets and the flow in the microfluidic channel. This enables the experimenter to intuitively evaluate the experimental state and quickly identify problems, such as inconsistent droplet sizes or air leakage, further enhancing the control precision of the experiment.

[0083] The single-cell sequencing library preparation instrument of the present application, through the comprehensive application of pressure monitoring and visual monitoring systems, can reduce experimental failures caused by equipment malfunctions or improper experimental conditions, ensure real-time quality control of the experiment during operation, enable users to terminate the experiment at any time when experimental abnormalities are found, will significantly improve the operation efficiency and data quality of droplet-based single-cell sequencing, and ensure the accuracy of experimental results. Repeated experiments and sample waste are common and costly problems in the prior art. The single-cell sequencing library preparation instrument of the present application can detect and solve problems in a timely manner, avoiding repeated experiments and sample waste. Precise monitoring also means reduced variability during data collection, improving the repeatability and reliability of the data. For critical applications such as gene expression analysis, this is particularly important because the accuracy of the data directly affects the interpretation of research results and subsequent applications.

[0084] The gas supply component and single-cell sequencing library preparation instrument of the present application improve the existing single-cell sequencing technology and achieve remarkable technical effects in aspects such as real-time quality control and experimental efficiency, which are specifically reflected in the following aspects:

[0085] (1) Improve operation flexibility: It allows users to adjust experimental settings according to real-time feedback, providing higher operation flexibility. Since it allows users to optimize experimental conditions to obtain the best results, it is particularly valuable for processing complex or variable biological samples.

[0086] (2) Save costs and resources: Real-time monitoring reduces repeated experiments and resource waste caused by experimental failures, especially when using precious or difficult-to-obtain samples, and the savings in costs and resources are particularly significant. In addition, increasing the success rate of experiments can accelerate the research progress and shorten the project timeline.

[0087] (3) Expand application fields: Due to the improved quality control and data reliability, it can expand the application of single-cell technology in clinical research and precision medicine. For example, it can be used for single-cell analysis during disease diagnosis, where the accuracy and reliability of the data are crucial.

[0088] In summary, the single-cell sequencing library construction instrument of the present application adopts the innovative integration of real-time pressure monitoring and visual monitoring systems, which not only significantly improves the operation 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 the single-cell sequencing library construction instrument of the present application solve a long-existing problem in droplet-based single-cell sequencing, namely how to achieve real-time quality control; by introducing an advanced monitoring system, it can detect and adjust experimental conditions in real time throughout the 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.

[0089] It should be noted that in this article, the term "including", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device including that element.

[0090] As described above, the above are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of changes or substitutions, which should be covered by the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to 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 component (500) includes a gas generator (510), 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). A chip pressure sensor (580) for detecting airway pressure is arranged on the outlet side of the pressure output module (570). The control valve (540) adjusts the gas pressure leading to the pressure output module (570) according to the pressure detection data of the chip pressure sensor (580).

2. The air supply assembly according to claim 1, characterized in that: The gas supply assembly (500) is also provided with a second gas generator (520) and a gas generator pressure sensor (530); 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; the gas generator pressure sensor (530) is connected to the gas path from the first three-way valve to the control valve (540).

3. The air supply assembly according to claim 1 or 2, characterized in that: A plurality of air branches are provided from the gas generator (510) to the pressure output module (570), each of the air branches is provided with a regulating valve (540), a corresponding number of air supply passages are provided in the pressure output module (570) corresponding to the air branches, and a chip pressure sensor (580) is provided on the outlet side of each of the air supply passages; the pressure output module (570) is provided with a plurality of air supply interfaces in series on each of the air supply passages, and each of the air supply interfaces is connected to a reaction hole of the chip (800).

4. The air supply assembly according to claim 3, characterized in that: A chip pressure sensor (580) is provided on the outlet side of each of the air supply passages of the pressure output module (570), and each of the control valves (540) adjusts the gas pressure of the air supply passage leading to the pressure output module (570) according to the pressure detection data of the chip pressure sensor (580) on the corresponding bronchus.

5. The air supply assembly according to claim 3, characterized in that: The air supply assembly (500) is also provided with a second regulating valve (550) and a regulating valve pressure sensor (560), wherein the regulating valve pressure sensor (560) is connected to the bronchial passage from each regulating valve (540) to the pressure output module (570), and the second regulating valve (550) is bypassed to the bronchial passage where each regulating valve (540) is located via a second three-way valve.

6. The air supply assembly according to claim 5, characterized in that: The control valve (540) and the second control valve (550) are proportional valves.

7. The air supply assembly according to claim 1, characterized in that: The gas supply assembly (500) is further provided with a gas generator pressure sensor (530), and the gas generator pressure sensor (530) is connected to the gas path from the gas generator (510) to the control valve (540); And / or, the gas supply assembly (500) is further provided with a control valve pressure sensor (560), and the control valve pressure sensor (560) is connected to the gas path from the control valve (540) to the pressure output module (570).

8. The air supply assembly according to claim 7, characterized in that: The gas supply assembly (500) is further provided with a second gas generator (520), and the gas generator (510) and the second gas generator (520) are connected to a gas path leading to the control valve (540) via a first three-way valve; And / or, the gas supply assembly (500) is further provided with a second regulating valve (550), and the second regulating valve (550) is connected to the gas path of the regulating valve (540) leading to the pressure output module (570) through a second three-way valve.

9. A single-cell sequencing library construction instrument, characterized in that: It comprises a shell (100), a chip transport tray (200), a control main board (400) and an air supply assembly (500) according to any one of claims 1 to 8, wherein the chip transport tray (200) is used to carry a chip (800) and is arranged in the shell (100) so as to be movable outward and inward, and the control main board (400) and the air supply assembly (500) are arranged in the shell (100); the control valve (540) and the chip pressure sensor (580) are connected to the control main board (400) by signal.

10. The single-cell sequencing library construction instrument according to claim 9, characterized in that: The single-cell sequencing library construction instrument further comprises a display module (300), wherein the display module (300) is arranged on the housing (100) and has a signal connection to the control main board (400).

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