An automated single-cell serial capture screening system and methods of use thereof

Abstract

The present application relates to the technical field of cell manipulation and analysis, and relates to an automatic single-cell continuous capture and screening system and a use method thereof.The system comprises a bias liquid storage module, a cell injection module, a microfluidic chip, a cell capture module, a cell screening module and an imaging system.The bias liquid storage module, the cell injection module, the cell screening module, the cell capture module and the microfluidic chip are connected.The bias liquid storage module is used to generate a bias flow.The cell injection module is used to provide cells.The microfluidic chip is used to capture and screen cells.The cell capture module is used to capture cells.The cell screening module is used to screen cells.The imaging system is used to obtain images of captured cells and visually analyze the images.The present application designs a microfluidic channel double-bias flow to stabilize cell flow sampling, cooperates with a high-speed electromagnetic valve to realize rapid control of a gas path on a cell capture and screening channel fluid, and further combines a high-speed camera to analyze cell positions in different regions in real time, so that automatic single-cell continuous capture and screening are realized.

Description

Technical Field

[0001] This invention relates to the field of cell analysis and manipulation technology, and in particular to an automated single-cell continuous capture and screening system and its usage method. Background Technology

[0002] Existing technologies present a significant contradiction between high-throughput sorting and high-quality imaging, generally failing to simultaneously meet the demands of high efficiency and high information content in single-cell sorting. The main existing technological approaches and their structural drawbacks are detailed below.

[0003] 1. Flow cytometry and its core high-speed sorting system:

[0004] This system can only acquire one-dimensional or two-dimensional physical parameters such as the overall fluorescence intensity and scattered light of the cell, and cannot provide crucial high-quality spatial information such as morphological features, subcellular structural localization, fluorescence distribution patterns, and organelle dynamics. Due to the lack of image information support, the system cannot effectively distinguish cells with similar total fluorescence but vastly different spatial distributions, severely limiting its application in fine sorting based on image phenotypes.

[0005] 2. Intelligent Image-Activated Cell Sorting System:

[0006] Because cells are constantly in high-speed motion (typically with flow rates exceeding 1 m / s), the imaging system can only use low-magnification objectives and short exposure times for imaging, severely limiting image resolution and information content, and making it impossible to achieve high-quality imaging at the organelle level. Furthermore, motion blur and defocusing further degrade image quality, restricting the system's application in high-precision subcellular classification and preventing it from completely replacing screening methods based on static, high-quality images.

[0007] 3. Robotic arm-assisted single-cell sorting system:

[0008] The sorting speed is extremely low, typically only a few to tens of cells per minute, which cannot meet the needs of large-scale screening. At the same time, the physical movement speed, positioning accuracy, and sample accessibility of the robotic arm constitute the fundamental bottleneck of system throughput. In addition, the open operating environment is prone to sample cross-contamination, culture medium evaporation, and decreased cell viability, which seriously affects the reproducibility and reliability of experimental results and makes it difficult to achieve automated and continuous process integration.

[0009] 4. Microfluidic single-cell capture and imaging system:

[0010] These devices focus on cell capture and imaging, lacking an effective and integrated screening module, making it impossible to perform subsequent physical separation and collection of identified target cells. Although they excel in imaging information, their functional limitations limit their applicability to analytical studies and make them unsuitable for experimental scenarios requiring cell sorting based on phenotype.

[0011] 5. Batch-type microfluidic sorting system:

[0012] The cell capture rate of this type of device is easily affected by flow rate and concentration, the system has low complexity and reliability, poor automation, the experimental procedure is lengthy and cumbersome, and its practicality is limited.

[0013] In conclusion, existing technologies exhibit a clear "technological roadmap paradox":

[0014] While robotic arm sorting and batch microfluidic sorting can achieve high-quality imaging, their sorting throughput is extremely low, or their functional modules are fragmented. Although flow cytometry and intelligent image sorting have high sorting throughput, their image information dimensions are limited and their resolution is low, which cannot meet the requirements for high-precision sorting. Summary of the Invention

[0015] This invention aims to at least solve one of the technical problems existing in related technologies. To this end, this invention provides an automated single-cell continuous capture and screening system and its method of use, achieving automated single-cell capture and screening into target channels.

[0016] This invention provides an automated single-cell continuous capture and screening system, comprising a first biased liquid storage module, a second biased liquid storage module, a cell injection module, a microfluidic chip, a cell capture module, a cell screening module, an imaging system, an air compression module, and a negative pressure module;

[0017] One end of the first biased liquid storage module is connected to the air compression module;

[0018] One end of the second biased liquid storage module is connected to the air compression module;

[0019] The other end of the first biased liquid storage module is connected to the microfluidic chip;

[0020] The other end of the second biased liquid storage module is connected to the microfluidic chip;

[0021] The cell injection module is connected to the microfluidic chip;

[0022] One end of the cell screening module is connected to the microfluidic chip;

[0023] One end of the cell capture module is connected to the microfluidic chip;

[0024] The other end of the cell screening module is connected to the air compression module.

[0025] The other end of the cell capture module is connected to the air compression module;

[0026] The negative pressure module and the cell capture module are connected;

[0027] The first biased liquid storage module is used to generate the first bias flow;

[0028] The second biased reservoir module is used to generate a second biased flow;

[0029] The cell injection module is used to supply cells to the microfluidic chip;

[0030] The microfluidic chip is used to achieve continuous single-cell capture and screening.

[0031] The cell capture module is used to capture cells;

[0032] The cell screening module is used to control the microfluidic chip to screen cells;

[0033] The air compression module is used to control the flow of cells and bias flow, as well as to control the cell capture module and cell screening module.

[0034] The negative pressure module is used to control the cell capture module to capture cells;

[0035] The imaging system is used to acquire images of captured cells and analyze the operating area of ​​the microfluidic chip.

[0036] According to the present invention, an automated single-cell continuous capture and screening system is provided, wherein the working area of ​​the microfluidic chip is planar, including: a first bias flow channel, a second bias flow channel, a cell flow channel, a capture channel, a screening channel, a target channel, and a waste channel;

[0037] The first end of the working area has a first bias flow channel, a second bias flow channel, and a cell flow channel.

[0038] The cell flow channel is located between the first bias flow channel and the second bias flow channel;

[0039] The inlet of the first bias flow channel is connected to the other end of the first bias liquid storage module;

[0040] The inlet of the second bias flow channel is connected to the other end of the second bias liquid storage module;

[0041] The cell flow channel is connected to the cell injection module;

[0042] The second end of the work area has a target passage and an abandoned passage;

[0043] The second end of the working area is opposite to the first end of the working area;

[0044] The target channel is used to collect screened cells;

[0045] The waste channel is used to collect waste cells;

[0046] The third end of the working area has a capture channel and a screening channel;

[0047] The capture channel is connected to one end of the cell capture module;

[0048] The screening channel is connected to one end of the cell screening module.

[0049] According to the present invention, an automated single-cell continuous capture and screening system is provided, wherein the working area includes a cell capture port for capturing cells;

[0050] The cell capture port is connected to the capture channel via a gap channel;

[0051] The gap channel can be a single channel, two parallel channels, or three parallel channels.

[0052] The gap channel is used to trap cells when they are drawn into the cell capture port.

[0053] According to the present invention, an automated single-cell continuous capture and screening system is provided, wherein the first biased liquid storage module includes a first pressure regulating valve and a first gas-push liquid storage tube;

[0054] The input end of the first pressure regulating valve is connected to the air compression module;

[0055] The output end of the first pressure regulating valve is connected to the air insertion end of the first pneumatic liquid storage tube;

[0056] The liquid insertion end of the first gas-pumped liquid storage tube is connected to the inlet of the first bias flow channel of the microfluidic chip;

[0057] The second biased liquid storage module includes a second pressure regulating valve and a second gas-push liquid storage tube;

[0058] The input end of the second pressure regulating valve is connected to the air compression module;

[0059] The output end of the second pressure regulating valve is connected to the air insertion end of the second air-push liquid storage tube;

[0060] The liquid insertion end of the second gas-push liquid storage tube is connected to the inlet of the second bias flow channel of the microfluidic chip.

[0061] According to the present invention, an automated single-cell continuous capture and screening system is provided, wherein the cell capture module includes a third pressure regulating valve, a fourth pressure regulating valve, a vacuum pressure regulating valve, a first three-way solenoid valve, a second three-way solenoid valve, a first height regulator, and a cell capture gas-push liquid storage tube;

[0062] The cell capture gas propulsion liquid storage tube, the first three-way solenoid valve and the second three-way solenoid valve are fixed on the first height adjuster.

[0063] The liquid insertion end of the cell-capturing gas-pumped liquid reservoir is connected to the capture channel inlet of the microfluidic chip;

[0064] The insertion gas end of the cell-capturing gas-pumped liquid reservoir is connected to the output end of the first three-way solenoid valve.

[0065] The normally closed input terminal of the first three-way solenoid valve is connected to the vacuum pressure valve.

[0066] The vacuum pressure valve is connected to the module;

[0067] The normally open input terminal of the first three-way solenoid valve is connected to the output terminal of the second three-way solenoid valve;

[0068] The normally closed input terminal of the second three-way solenoid valve is connected to the third pressure regulating valve;

[0069] The normally open input terminal of the second three-way solenoid valve is connected to the fourth pressure regulating valve;

[0070] One end of the third pressure regulating valve is connected to the air compression module;

[0071] One end of the fourth pressure regulating valve is connected to the air compression module.

[0072] According to the present invention, an automated single-cell continuous capture and screening system is provided, wherein the cell screening module includes: a second height regulator, a cell screening gas-push liquid reservoir, a third three-way solenoid valve, a fifth pressure regulating valve, and a sixth pressure regulating valve;

[0073] The cell screening gas-pumped liquid storage tube and the third three-way solenoid valve are fixed on the second height regulator.

[0074] The liquid insertion end of the cell screening gas-pumped liquid reservoir is connected to the screening channel outlet of the microfluidic chip.

[0075] The gas insertion end of the cell screening gas-pumped liquid reservoir is connected to the output end of the third three-way solenoid valve.

[0076] The normally closed input terminal of the third three-way solenoid valve is connected to the fifth pressure regulating valve.

[0077] The normally closed input terminal of the third three-way solenoid valve is connected to the sixth pressure regulating valve.

[0078] One end of the fifth pressure regulating valve is connected to the air compression module;

[0079] One end of the sixth pressure regulating valve is connected to the air compression module.

[0080] According to the present invention, an automated single-cell continuous capture and screening system is provided, wherein the imaging system includes an upright optical path and an inverted optical path;

[0081] The upright optical path includes an LED light source, a beam splitter, a long-pass filter, a first objective lens, and an ultrafast camera;

[0082] The light emitted by the LED light source passes through a beam splitter, enters the first objective lens, and then reaches the observation area of ​​the microfluidic chip.

[0083] After receiving reflected light from the microfluidic working area, the first objective lens passes through a beam splitter and then through a long-pass filter that only allows light with wavelengths greater than or equal to the LED light source to enter the ultrafast camera.

[0084] The inverted optical path is an arbitrary inverted microscopic imaging system.

[0085] The present invention also provides a method of using an automated single-cell continuous capture and screening system, comprising:

[0086] S100: Activate the first bias reservoir module, the second bias reservoir module, and the cell injection module to form a stable flow line for the cell suspension. Under the guidance of the bias flow, the suspension flows to the waste channel. Adjust the resting pressure parameters of the cell capture module and the cell screening module so that the cells flowing through the cell capture port are not passively captured or enter the screening channel in advance. Instead, they smoothly enter the waste channel with the main liquid flow. Furthermore, the cells are not passively pushed out of the cell capture port after being actively captured.

[0087] S200: Utilizes the upright optical path of the imaging system to continuously acquire video streams of the working area. By analyzing the cell morphology, size and number in different ROIs, when a suitable target cell is found, the host computer sends a pulse signal to draw in the target cell and fix it at the cell capture port.

[0088] S300: For the captured single cell, start the inverted optical path of the imaging system to perform high-quality imaging; the host computer determines whether the cell is the target phenotype based on image analysis: if not, the host computer sends a pulse signal to control the opening and closing of the second three-way solenoid valve to release the captured cell, allowing it to flow into the waste channel along the initial cell flow path, and returns to step S200; if yes, then execute step S400.

[0089] S400: The host computer sends a pulse signal to control the opening and closing of the second three-way high-speed solenoid valve to release the captured cells. Then, it controls the opening and closing of the third three-way high-speed solenoid valve of the cell screening module to push the released target cells into the target cell channel, thus completing the sorting of the captured target cells.

[0090] The present invention provides a method for using an automated single-cell continuous capture and screening system, controlled by a multi-ROI machine vision algorithm, specifically including:

[0091] S10: Continuous Image Acquisition and Analysis: The ultrafast camera acquires images of the working area in real time, and the host computer processes them in parallel through multi-threading and analyzes the preset ROIs in real time, including the cell flow ROI, the capture ROI, the cell capture port ROI, and the screening ROI.

[0092] S20: Condition-triggered capture decision: The capture command is triggered only when the following conditions are met simultaneously within the capture ROI: (a) there is a cell whose shape and size conform to the preset parameters; (b) the cell flows through the ROI without interference from other cells;

[0093] S30: Capture Verification and Fault Tolerance: After triggering the capture command, delay for 20ms to 100ms, and then analyze the cell capture port ROI; if the cells are confirmed to be successfully fixed, proceed to the imaging judgment in step S300; if the capture fails, return to S200 to continue monitoring;

[0094] S40: Precise control of screening timing: For target cells, the release and sorting actions must meet the following requirements: Before release, there are no cells in the ROI when the cells flow through it; after release, the target cells must appear in the screening ROI within the set time window before the screening pulse is triggered, ensuring the uniqueness and accuracy of the sorting action. After screening, the collection ROI located in the cell collection channel is analyzed in real time within 50ms. The appearance of cells indicates that the cell screening is successful.

[0095] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0096] The automated continuous single-cell capture and screening system and its usage method provided by this invention have the following advantages:

[0097] 1. An active single-cell capture and sorting device that significantly improves cell capture efficiency compared to passive cell capture microfluidic devices, and also enables the sorting of target cells after capture:

[0098] The innovative microfluidic channel design features dual-biased flow to stabilize cell flow, combined with capture and screening channels. High-speed solenoid valves provide millisecond-level pulsed negative and positive pressure fluid control, enabling active single-cell capture and screening into the target channel. Compared to traditional passive fluid capture chips, this design offers higher cell capture efficiency and can push captured and released target cells into the target channel, thus completing the target cell screening function.

[0099] Precise and efficient cell capture and sorting: Based on real-time cell analysis of multiple ROI machine vision using an ultrafast camera (greater than 1000FPS), combined with fluid control with high-speed solenoid valves for rapid response, automated, fast, and precise continuous capture and sorting of single cells is achieved.

[0100] 2. It achieves the integration of high-throughput, high-quality imaging with full-process automation, breaking through the performance bottlenecks of existing technologies:

[0101] High-throughput continuous operation: It overcomes the shortcomings of robotic arm sorting (several cells per minute) and batch microfluidic technology (>20 seconds / cell) with extremely low throughput, and realizes full automation and seamless connection of the "capture-imaging-sorting" process. The total time for single cell capture-release and screening is only about 100ms, and the throughput is increased by more than 100 times.

[0102] High-quality imaging screening: This method overcomes the limitation of not being able to obtain high-quality cell image information under high-speed cell flow conditions such as flow cytometry. After capturing and fixing single cells, bright-field or fluorescence imaging of cells can be performed using a high-power objective lens, and then cell screening can be performed based on the imaging results. This provides a platform for sorting and screening based on high-quality cell images.

[0103] High reliability and cell viability: The fully enclosed operation avoids the problems of sample contamination, evaporation and decreased cell viability caused by open environments (such as robotic arms) or frequent cleaning (such as batch microfluidics), and the repeatability and reliability of experimental results are fundamentally improved.

[0104] 3. The system has a streamlined structure, significantly reduced costs, and high practicality and accessibility:

[0105] Hardware cost advantage: Compared with high-end equipment that relies on expensive robotic arms or complex droplet sorting systems, the core control of this invention is based on conventional high-speed solenoid valves and pressure regulating valves, which greatly reduces hardware costs.

[0106] Innovative control scheme: Through innovative air circuit series design and height regulator for fine-tuning pressure, a simple and reliable solution is achieved to achieve precision fluid control comparable to high-end equipment, solving the engineering problem that pressure reducing valves alone cannot meet the requirements of fine liquid pressure control in microfluidic chips.

[0107] High applicability: It significantly lowers the barrier to entry for high-performance single-cell sorting technology, enabling ordinary laboratories to conduct high-throughput, high-content single-cell sorting research, and greatly promotes the application of this technology.

[0108] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0109] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0110] Figure 1 This is a schematic diagram of the structure of the present invention.

[0111] Figure 2 This is a schematic diagram of the microfluidic chip provided by the present invention.

[0112] Figure 3 This is the optical path diagram of the imaging system of the present invention.

[0113] Figure 4 This is a schematic diagram of the gas-pumped fluid structure provided by the present invention.

[0114] Figure 5 This is a complete structural schematic diagram of the present invention.

[0115] Figure 6 This is a flowchart illustrating the single-cell capture, release, and screening process in the working area of ​​the microfluidic chip of this invention.

[0116] Figure 7 This is a working diagram of single-cell capture and target cell screening.

[0117] Figure 8 This is a flowchart of the real-time machine vision cell recognition algorithm based on background subtraction proposed in this invention.

[0118] Figure 9 This is a flowchart of the method of using the device proposed in this invention.

[0119] Figure label:

[0120] 4. Microfluidic chip working area; 7. Channel outlet; 9. Imaging system; 10. Air compression module; 41. First bias flow channel; 42. Second bias flow channel; 43. Cell flow channel; 44. Capture channel; 45. Screening channel; 46. Target channel; 47. Waste channel; 48. Gap channel; 49. Cell capture port; 91. LED light source; 92. Beam splitter; 93. Long-pass filter; 94. First objective lens; 95. Ultrafast camera; 96. Inverted imaging system; 101. PU tube; 102. Microfluidic capillary; 103. 104. Cardiac tube sealing bayonet; 11. Liquid storage tube; 12. First pressure regulating valve; 20. First pneumatic liquid storage tube; 21. Host computer and control module; 22. Second pressure regulating valve; 31. Injection pump; 32. Single-cell sampler; 51. Third pressure regulating valve; 52. Fourth pressure regulating valve; 53. Vacuum pressure regulating valve; 54. First three-way solenoid valve; 55. Second three-way solenoid valve; 56. Negative pressure module; 57. First height adjuster; 61. Second height adjuster; 62. Third three-way solenoid valve; 63. Fifth pressure regulating valve; 64. Sixth pressure regulating valve. Detailed Implementation

[0121] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention but cannot be used to limit the scope of this invention.

[0122] In the description of the embodiments of the present invention, it should be noted that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of the present invention. In addition, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0123] In the description of the embodiments of the present invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "connected" and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of the present invention based on the specific circumstances.

[0124] In embodiments of the present invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0125] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0126] The following is combined Figures 1 to 9 This invention is described.

[0127] Example

[0128] like Figure 1 As shown, Figure 1 This is a schematic diagram of a microfluidic chip structure for an automated single-cell continuous capture and screening system, including:

[0129] It consists of a first bias flow channel 41, a second bias flow channel 42, a cell flow channel 43, a microfluidic chip working area 4, a capture channel, a screening channel, a target channel outlet, and a waste channel outlet;

[0130] The first bias flow channel 41 and the second bias flow channel 42 are used to introduce a bias flow to stabilize the flow field and force the cell solution into the waste channel;

[0131] The cell flow channel is used to introduce a single-cell suspension;

[0132] The capture channel 44 is used to capture single cells when negative pressure is input, and to release the captured single cells when positive pressure is input.

[0133] When a positive pressure is applied to the screening channel 45, the target cells released from the cell capture port 49 are pushed into the target channel.

[0134] The target channel is used to collect the screened target cells;

[0135] The waste channel 47 is used to discharge uncaptured cells or released non-target cells;

[0136] The working area 4 of the microfluidic chip is the working area for single-cell capture and screening, and its enlarged structural diagram is shown below. Figure 2 As shown, it includes:

[0137] First bias flow channel 41, second bias flow channel 42, cell flow channel 43, capture channel 44, screening channel 45, target channel 46, waste channel 47, gap channel 48, cell capture port 49;

[0138] The biased flow of the first biased flow channel 41 and the second biased flow channel 42 stabilizes the cell solution flowing out of the cell flow channel 43 on the side close to the cell capture port 49 and flows out from the waste channel 47.

[0139] The cell capture port 49 is connected to the capture channel 44 via the gap channel 48. When the capture channel is under negative pressure, the cell is drawn into the cell capture port 49 and stuck through the channel gap.

[0140] The capture channel 44 captures cells under negative pressure and releases captured cells under slightly positive pressure.

[0141] When the screening channel 45 is under positive pressure, the released target captured cells are pushed into the target channel 46.

[0142] Specifically, such as Figure 3 As shown, the imaging system 9 includes an upright optical path and an inverted optical path;

[0143] The upright optical path is used to provide real-time images for machine vision, supporting high-speed cell capture and sorting decisions; the inverted optical path is used to image the captured single cells, providing a basis for sorting.

[0144] The upright optical path includes an LED light source 91, a beam splitter 92, a long-pass filter 93, a first objective lens 94, and an ultrafast camera 95;

[0145] The light emitted by the LED light source 91 passes through the beam splitter 92, enters the first objective lens 94, and then reaches the working area 4 of the microfluidic chip for illumination.

[0146] After receiving the illumination reflected light from the working area 4 of the microfluidic chip, the first objective lens 94 passes through the beam splitter 92 and then through the long-pass filter 93, which only allows wavelengths greater than or equal to the LED light source to pass through, into the ultrafast camera 95.

[0147] The ultrafast camera 95 acquires images of the working area 4 of the microfluidic chip in real time and performs ROI analysis to provide decisions for single-cell capture and sorting.

[0148] The inverted imaging system 96 can be any inverted microscopic imaging system, which performs high-quality imaging of single cells at the cell capture port after capture, and determines whether it is a target cell through a host computer.

[0149] Specifically, such as Figure 4 As shown, Figure 4 This is a schematic diagram of the pneumatic fluid structure provided by the present invention, wherein the first pneumatic fluid storage tube 12, the second pneumatic fluid storage tube 22, the pneumatic fluid storage tube in the first height adjuster 57, and the storage tube in the second height adjuster 61 are all... Figure 4 The gas-pumped liquid structure described in the text:

[0150] The reagent solution is stored in a 0.5 mL reservoir tube 104. The centrifuge tube sealing bayonet 103 is sealed with a PDMS plug with two holes to ensure that the air content is less than 0.1 mL, thereby reducing the pneumatic balance time after the solenoid valve is activated and improving the fluid control response speed of the system.

[0151] Two holes are inserted into a PU (Polyurethane) gas tube and a microfluidic capillary tube 102, respectively, and externally secured with a 3D-printed centrifuge tube sealing bayonet 103 to prevent high-pressure gas from causing the plug to shift and making the fluid unstable.

[0152] One end of the microfluidic capillary 102 is inserted into the liquid surface, and the other end is connected to the microfluidic chip;

[0153] The PU tube 101 is not immersed below the liquid surface. Its other end is connected to the gas circuit control system. By inputting different air pressures, fluid control within the microfluidic chip can be achieved, thereby enabling the capture, release, and screening of cells within the microfluidic chip.

[0154] Specifically, such as Figure 5 The diagram shows the complete structure of the present invention.

[0155] The first bias flow module consists of a first pressure regulating valve 11 and a first air-push liquid storage pipe 12 to provide a continuous and stable positive pressure to drive the liquid to flow smoothly.

[0156] The second bias flow module consists of a second pressure regulating valve 21 and a second gas-push liquid storage pipe 22 to provide a continuous and stable positive pressure to drive the liquid to flow smoothly.

[0157] The cell injection module consists of an injection pump 31 and a single-cell sampler 32, used to generate a stable flow of single cells.

[0158] The output end of the injection pump 31 is connected to the input end of the single-cell sampler 32;

[0159] The output end of the single-cell sampler 32 is connected to the cell flow channel 43;

[0160] The single-cell sampler 32 uses an injection pump in conjunction with mineral oil with a density lower than water to inject the cell suspension via an oil-driven water mechanism.

[0161] The specific steps are as follows:

[0162] The microsyringe is pre-filled with mineral oil and connected to a single-hole silicone stopper via a microfluidic capillary. This stopper is then sealed to the tip of a 10μL pipette.

[0163] When loading the sample, the syringe pump first pushes out mineral oil to fill the pipette tip, then the pipette tip is immersed in the single-cell suspension, and the syringe pump is pulled back to draw in about 20 μL of cell sample.

[0164] Once completed, insert the pipette tip into the cell flow channel 43 of the chip to achieve sample loading without dead volume.

[0165] This method significantly reduces sample waste and is suitable for small quantities of rare cell samples. The use of an injection pump also allows for precise control of the perfusion flow rate of the cell suspension.

[0166] The cell capture module consists of a first height regulator 57, a pneumatic liquid reservoir, a first three-way solenoid valve 54, a second three-way solenoid valve 55, a vacuum pressure valve 53, a third pressure regulating valve 51, and a fourth pressure regulating valve 52, and is used to capture and release captured single cells.

[0167] The cell screening module consists of a second height regulator 61, a pneumatic liquid reservoir, a third three-way solenoid valve 62, a fifth pressure regulating valve 63, and a sixth pressure regulating valve 64, and is used to push target cells into the target channel 46.

[0168] The microfluidic capillary 102 of the gas-pumped liquid reservoir tube of the cell capture module is connected to the capture channel 44 of the microfluidic chip, and the PU gas tube 101 end of the gas-pumped liquid reservoir tube is connected to the output end of the first three-way solenoid valve 54.

[0169] The normally closed air inlet of the first three-way solenoid valve 54 of the cell capture module is connected to the output end of the vacuum pressure valve 53, and the normally open air inlet is connected to the output end of the second three-way solenoid valve 55.

[0170] The normally closed air inlet of the second three-way solenoid valve 55 of the cell capture module is connected to the output end of the third pressure regulating valve, and the normally open air inlet is connected to the output end of the fourth pressure regulating valve 52.

[0171] The input end of the vacuum pressure valve 53 of the cell capture module is connected to the negative pressure module 56;

[0172] The input terminals of the third pressure regulating valve 51 and the fourth pressure regulating valve 52 of the cell capture module are connected to the air compression module 10.

[0173] The microfluidic capillary 102 of the gas-pumped liquid reservoir tube of the cell screening module is connected to the screening channel 45 of the microfluidic chip, and the PU gas tube 101 end of the gas-pumped liquid reservoir tube is connected to the output end of the third three-way solenoid valve 62.

[0174] The normally closed air inlet of the third three-way solenoid valve 62 of the cell screening module is connected to the output end of the fifth pressure regulating valve 63, and the normally open air inlet is connected to the output end of the sixth pressure regulating valve 64.

[0175] The input terminals of the fifth pressure regulating valve 63 and the sixth pressure regulating valve 64 of the cell capture module are connected to the air compression module 10.

[0176] The power control of all the three-way solenoid valves is connected to the host computer and control module 20. The instantaneous air pressure switching is achieved through valve control to perform cell capture, release and screening operations.

[0177] The air pressure output by the first height adjuster 57 and the fourth pressure regulating valve 52, which is connected to the normally open end of the three-way high-speed solenoid valve, jointly regulates the air pressure of the capture channel, so that there is a slight pressure difference between the cell capture port 49 and the capture channel 44, so that the cells are not passively sucked into the cell capture port 49, and the captured cells can be fixed in the cell capture port 49.

[0178] The air pressure output by the second height regulator 61 and the sixth pressure regulating valve 64, which is connected to the normally open end of the three-way high-speed solenoid valve, jointly regulates the air pressure of the screening channel, so that the screening channel 45 exhibits a slight liquid inflow into the waste channel 47, thereby preventing cells from flowing into the screening channel and also preventing the flowing cells from being passively pushed into the target channel 46.

[0179] The negative pressure module 56 is connected to the cell capture module and provides negative pressure for capturing cells;

[0180] The air compression module 10 is used to control the flow of the bias flow and to control the cell capture module to adjust the micro pressure and release the captured cells and the cell screening module to screen target cells.

[0181] The embodiments of the present invention also include a host computer and a control module 20, used to control the start-up time of the solenoid valve.

[0182] Specifically, such as Figure 6 As shown, Figure 6 This is a flowchart illustrating the single-cell capture, release, and screening process in the working area of ​​the microfluidic chip of this invention.

[0183] like Figure 6 As shown in (a), under the action of the first bias flow and the second bias flow, the cells in the resting state flow steadily into the waste channel;

[0184] Figure 6 As shown in (b), when a cell flows through an ROI (Region of Interest) (solid box in the figure) and there is only one cell in its subset "Capture ROI" (dashed box in the figure), the pulse activates the first three-way solenoid valve, and the capture channel generates a pulse negative pressure, which draws the cell into the cell capture port.

[0185] Figure 6 As shown in (c), the ROI region of the cell capture port is analyzed. When a cell is successfully captured, imaging is performed and the results are analyzed to determine whether it is the target cell. A magnified cross-sectional view of the cell capture port shows the state of the cell after capture: because the interstitial channel 48 is only 5 μm high, the cell (typically 10-20 μm in diameter) undergoes partial deformation and becomes stuck at the capture port, as shown in (c). Figure 6 As shown in (f), the capture of cells was fixed.

[0186] Figure 6 As shown in (d), when the captured cells are the target screening cells, in order to prevent false screening, the ROI of the cell flow area needs to be analyzed in real time. Only when there are no cells in the ROI is the second three-way solenoid valve activated by a pulse. The capture channel generates a pulse positive pressure to push the cells at the capture port into the cell flow, thus completing the release of the captured cells.

[0187] Figure 6 As shown in (e), when releasing cells as target cells, the ROI of the screening area is detected. When cells appear, the third three-way solenoid valve is activated by a pulse. The pulsed water flow in the screening channel pushes the target cells above the channel into the target channel. At the same time, the ROI of the collection area is detected. The appearance of cells in the collection area indicates that the screening of target cells has been successfully achieved.

[0188] Figure 7 (a) and Figure 7 (b) shows actual working images of single-cell capture and target cell screening, respectively.

[0189] The entire cell capture, release, and screening process is based on real-time image cell recognition of multiple preset Regions of Interest (ROIs) using an ultrafast camera. By adjusting the flow rate of the syringe pump to control cell movement speed, and by optimizing the ROI position and size, as well as adjusting the activation time of the solenoid valve, the success rate of capture and screening can be significantly improved. In the actual test, a concentration of approximately 1×10⁻⁶ was used. 6 HeLa single-cell suspensions at a flow rate of 0.15 μL / min were perfused (apparent cell flow rate approximately 7 μm / ms). One second was reserved after each capture for imaging and evaluation. During the experiment, which lasted 2027 seconds, 1408 captures were completed, achieving a capture success rate of 94.38%; of these, 1343 were successfully screened, resulting in a screening success rate of 95.16%. Based on this microfluidic system, the average manipulation cycle per cell was 0.51 seconds. The time from cell capture to fluid stabilization after cell capture is approximately 30 ms, the time for cell release and sorting is approximately 70 ms, and the total time from cell capture to completion of sorting is approximately 100 ms. On average, about 0.4 ms of the 0.51 ms is spent waiting for the target cells to flow through during the cell capture step. The overall sorting speed is as high as 117 cells / min. By increasing the cell density of the cell solution and reducing the waiting time during cell capture, the average manipulation time of a single cell can be further reduced, achieving sub-second high-throughput, continuous single-cell capture, release, and sorting.

[0190] like Figure 8 As shown, Figure 8 This is a flowchart of the real-time machine vision cell recognition algorithm based on background subtraction proposed in this invention. It includes the following steps:

[0191] S1000: Acquires a background image and performs Gaussian blur on the background image to obtain a preprocessed image;

[0192] S2000: Obtain a grayscale image by performing grayscale difference analysis on the preprocessed image;

[0193] S3000: Binarize the grayscale image to obtain a binary image;

[0194] S4000: After performing morphological calculations on the binarized image to remove small particles and cavities, the outline is identified, the number of cells is calculated, and whether it is a target cell is determined based on the area and roundness.

[0195] Specifically, such as Figure 9 As shown, the present invention also provides a method of using an automated single-cell continuous capture and screening system, comprising:

[0196] S100: Activate the first bias reservoir module, the second bias reservoir module, and the cell injection module to form a stable flow line for the cell suspension. Under the guidance of the bias flow, the suspension flows to the waste channel. Adjust the resting pressure parameters of the cell capture module and the cell screening module so that the cells flowing through the cell capture port are not passively captured or enter the screening channel in advance. Instead, they smoothly enter the waste channel with the main liquid flow. Furthermore, the cells are not passively pushed out of the cell capture port after being actively captured.

[0197] S200: Utilizes the upright optical path of the imaging system to continuously acquire video streams of the working area. By analyzing the cell morphology, size and number in different ROIs, when a suitable target cell is found, the host computer sends a pulse signal to draw in the target cell and fix it at the cell capture port.

[0198] S300: For the captured single cell, start the inverted optical path of the imaging system to perform high-quality imaging; the host computer determines whether the cell is the target phenotype based on image analysis: if not, the host computer sends a pulse signal to control the opening and closing of the second three-way solenoid valve to release the captured cell, allowing it to flow into the waste channel along the initial cell flow path, and returns to step S200; if yes, then execute step S400.

[0199] S400: The host computer sends a pulse signal to control the opening and closing of the second three-way high-speed solenoid valve to release the captured cells. Then, it controls the opening and closing of the third three-way high-speed solenoid valve of the cell screening module to push the released target cells into the target cell channel, thus completing the sorting of the captured target cells.

[0200] Specifically, the automated loop of steps S200 to S400 is controlled by a multi-ROI-based machine vision algorithm, which includes:

[0201] S10: Continuous Image Acquisition and Analysis: The ultrafast camera acquires images of the working area in real time, and the host computer processes them in parallel through multi-threading and analyzes the preset ROIs in real time, including the cell flow ROI, the capture ROI, the cell capture port ROI, and the screening ROI.

[0202] S20: Condition-triggered capture decision: The capture command is triggered only when the following conditions are met simultaneously within the capture ROI: (a) there is a cell whose shape and size conform to the preset parameters; (b) the cell flows through the ROI without interference from other cells;

[0203] S30: Capture Verification and Fault Tolerance: After triggering the capture command, delay for 20ms to 100ms, and then analyze the cell capture port ROI; if the cells are confirmed to be successfully fixed, proceed to the imaging judgment in step S300; if the capture fails, return to S200 to continue monitoring;

[0204] S40: Precise control of screening timing: For target cells, the release and sorting actions must meet the following requirements: Before release, there are no cells in the ROI when the cells flow through it; after release, the target cells must appear in the screening ROI within the set time window before the screening pulse is triggered, ensuring the uniqueness and accuracy of the sorting action. After screening, the collection ROI located in the cell collection channel is analyzed in real time within 50ms. The appearance of cells indicates that the cell screening is successful.

[0205] Furthermore, although the operation of the methods of this disclosure is described in a specific order in the accompanying drawings, this does not require or imply that these operations must be performed in that specific order, or that all the operations shown must be performed to achieve the desired result. Rather, the steps depicted in the flowcharts may be performed in a different order. Additionally or alternatively, certain steps may be omitted, multiple steps may be combined into one step, and / or one step may be broken down into multiple steps. It should also be noted that the features and functions of two or more devices according to this disclosure may be embodied in one device. Conversely, the features and functions of one device described above may be further divided and embodied by multiple devices.

[0206] While this disclosure has been described with reference to several specific embodiments, it should be understood that this disclosure is not limited to the specific embodiments disclosed. This disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. An automated single-cell continuous capture and screening system, characterized in that, include: The system comprises a first biased liquid reservoir module, a second biased liquid reservoir module, a cell injection module, a microfluidic chip, a cell capture module, a cell screening module, an imaging system, an air compression module, and a negative pressure module. One end of the first biased liquid storage module is connected to the air compression module; One end of the second biased liquid storage module is connected to the air compression module; The other end of the first biased liquid storage module is connected to the microfluidic chip; The other end of the second biased liquid storage module is connected to the microfluidic chip; The cell injection module is connected to the microfluidic chip; One end of the cell screening module is connected to the microfluidic chip; One end of the cell capture module is connected to the microfluidic chip; The other end of the cell screening module is connected to the air compression module. The other end of the cell capture module is connected to the air compression module; The negative pressure module and the cell capture module are connected; The first biased liquid storage module is used to generate the first bias flow; The second biased reservoir module is used to generate a second biased flow; The cell injection module is used to supply cells to the microfluidic chip; The microfluidic chip is used to achieve continuous single-cell capture and screening. The cell capture module is used to capture cells; The cell screening module is used to control the microfluidic chip to screen cells; The air compression module is used to control the flow of cells and bias flow, as well as to control the cell capture module and cell screening module. The negative pressure module is used to control the cell capture module to capture cells; The imaging system is used to acquire images of captured cells and analyze the operating area of ​​the microfluidic chip; The usage method includes the following steps: S100: Activate the first bias reservoir module, the second bias reservoir module, and the cell injection module to form a stable flow line for the cell suspension. Under the guidance of the bias flow, the suspension flows to the waste channel. Adjust the resting pressure parameters of the cell capture module and the cell screening module so that the cells flowing through the cell capture port are not passively captured or enter the screening channel in advance. Instead, they smoothly enter the waste channel with the main liquid flow. Furthermore, the cells are not passively pushed out of the cell capture port after being actively captured. S200: Utilizes the upright optical path of the imaging system to continuously acquire video streams of the working area. By analyzing the cell morphology, size and number in different ROIs, when a suitable target cell is found, the host computer sends a pulse signal to draw in the target cell and fix it at the cell capture port. S300: For the captured single cell, start the inverted optical path of the imaging system to perform high-quality imaging; the host computer determines whether the cell is the target phenotype based on image analysis: if not, the host computer sends a pulse signal to control the opening and closing of the second three-way solenoid valve to release the captured cell, allowing it to flow into the waste channel along the initial cell flow path, and returns to step S200; if yes, then execute step S400. S400: The host computer sends a pulse signal to control the opening and closing of the second three-way high-speed solenoid valve to release the captured cells. Then, it controls the opening and closing of the third three-way high-speed solenoid valve of the cell screening module to push the released target cells into the target cell channel, thus completing the sorting of the captured target cells.

2. The automated single-cell continuous capture and screening system according to claim 1, characterized in that, The working area of ​​the microfluidic chip is planar, and channels are opened at three ends of the working area, including: a first bias flow channel, a second bias flow channel, a cell flow channel, a capture channel, a screening channel, a target channel, and a waste channel; The first end of the working area has a first bias flow channel, a second bias flow channel, and a cell flow channel. The cell flow channel is located between the first bias flow channel and the second bias flow channel; The inlet of the first bias flow channel is connected to the other end of the first bias liquid storage module; The inlet of the second bias flow channel is connected to the other end of the second bias liquid storage module; The cell flow channel is connected to the cell injection module; The second end of the work area has a target passage and an abandoned passage; The second end of the working area is opposite to the first end of the working area; The target channel is used to collect screened cells; The waste channel is used to collect waste cells; The third end of the working area has a capture channel and a screening channel; The capture channel is connected to one end of the cell capture module; The screening channel is connected to one end of the cell screening module.

3. The automated single-cell continuous capture and screening system according to claim 2, characterized in that, The working area includes a cell capture port for capturing cells; The cell capture port is connected to the capture channel via a gap channel; The gap channel can be a single channel, two parallel channels, or three parallel channels; The gap channel is used to trap cells when they are drawn into the cell capture port.

4. The automated single-cell continuous capture and screening system according to claim 1, characterized in that, The first biased liquid storage module includes a first pressure regulating valve and a first pneumatic liquid storage tube; The input end of the first pressure regulating valve is connected to the air compression module; The output end of the first pressure regulating valve is connected to the air insertion end of the first pneumatic liquid storage tube; The liquid insertion end of the first gas-pumped liquid storage tube is connected to the inlet of the first bias flow channel of the microfluidic chip; The second biased liquid storage module includes a second pressure regulating valve and a second gas-push liquid storage tube; The input end of the second pressure regulating valve is connected to the air compression module; The output end of the second pressure regulating valve is connected to the air insertion end of the second air-push liquid storage tube; The liquid insertion end of the second gas-push liquid storage tube is connected to the inlet of the second bias flow channel of the microfluidic chip.

5. The automated single-cell continuous capture and screening system according to claim 1, characterized in that, The cell capture module includes a third pressure regulating valve, a fourth pressure regulating valve, a vacuum pressure regulating valve, a first three-way solenoid valve, a second three-way solenoid valve, a first height adjuster, and a cell capture gas-pump liquid reservoir. The cell capture gas propulsion liquid storage tube, the first three-way solenoid valve and the second three-way solenoid valve are fixed on the first height adjuster. The liquid insertion end of the cell-capturing gas-pumped liquid reservoir is connected to the capture channel inlet of the microfluidic chip; The insertion gas end of the cell-capturing gas-pumped liquid reservoir is connected to the output end of the first three-way solenoid valve. The normally closed input terminal of the first three-way solenoid valve is connected to the vacuum pressure valve. The vacuum pressure valve is connected to the module; The normally open input terminal of the first three-way solenoid valve is connected to the output terminal of the second three-way solenoid valve; The normally closed input terminal of the second three-way solenoid valve is connected to the third pressure regulating valve; The normally open input terminal of the second three-way solenoid valve is connected to the fourth pressure regulating valve; One end of the third pressure regulating valve is connected to the air compression module; One end of the fourth pressure regulating valve is connected to the air compression module.

6. The automated single-cell continuous capture and screening system according to claim 1, characterized in that, The cell screening module includes: a second height regulator, a cell screening gas-pumped liquid reservoir, a third three-way solenoid valve, a fifth pressure regulating valve, and a sixth pressure regulating valve; The cell screening gas-pumped liquid storage tube and the third three-way solenoid valve are fixed on the second height regulator. The liquid insertion end of the cell screening gas-pumped liquid reservoir is connected to the screening channel outlet of the microfluidic chip. The gas insertion end of the cell screening gas-pumped liquid reservoir is connected to the output end of the third three-way solenoid valve. The normally closed input terminal of the third three-way solenoid valve is connected to the fifth pressure regulating valve. The normally closed input terminal of the third three-way solenoid valve is connected to the sixth pressure regulating valve. One end of the fifth pressure regulating valve is connected to the air compression module; One end of the sixth pressure regulating valve is connected to the air compression module.

7. The automated single-cell continuous capture and screening system according to claim 1, characterized in that, The imaging system includes an upright optical path and an inverted optical path; The upright optical path includes an LED light source, a beam splitter, a long-pass filter, a first objective lens, and an ultrafast camera; The light emitted by the LED light source passes through a beam splitter, enters the first objective lens, and then reaches the observation area of ​​the microfluidic chip. After receiving reflected light from the microfluidic working area, the first objective lens passes through a beam splitter and then through a long-pass filter that only allows light with wavelengths greater than or equal to the LED light source to enter the ultrafast camera. The inverted optical path is an arbitrary inverted microscopic imaging system.

8. The method of using the automated single-cell continuous capture and screening system according to claim 1, characterized in that, Machine vision algorithm control based on multiple ROIs specifically includes: S10: Continuous Image Acquisition and Analysis: The ultrafast camera acquires images of the working area in real time, and the host computer processes them in parallel through multi-threading and analyzes the preset ROIs in real time, including the cell flow ROI, the capture ROI, the cell capture port ROI, and the screening ROI. S20: Condition-triggered capture decision: The capture command is triggered only when the following conditions are met simultaneously within the capture ROI: (a) there is a cell whose shape and size conform to the preset parameters; (b) the cell flows through the ROI without interference from other cells; S30: Capture Verification and Fault Tolerance: After triggering the capture command, delay for 20ms to 100ms, and then analyze the cell capture port ROI; if the cells are confirmed to be successfully fixed, proceed to the imaging judgment in step S300; if the capture fails, return to S200 to continue monitoring; S40: Precise control of screening timing: For target cells, the release and sorting actions must meet the following requirements: Before release, there are no cells in the ROI when the cells flow through it; after release, the target cells must appear in the screening ROI within the set time window before the screening pulse is triggered, ensuring the uniqueness and accuracy of the sorting action. After screening, the collection ROI located in the cell collection channel is analyzed in real time within 50ms. The appearance of cells indicates that the cell screening is successful.

Images