Immune cell chemical staining instrument and use method thereof

Abstract

The invention relates to the field of immune cell chemistry, and discloses an immune cell chemical staining instrument and a use method thereof.The immune cell chemical staining instrument comprises a workbench, a sample bearing unit, a temperature control unit, a liquid treatment unit and a dynamic uniform mixing unit, and cover glass in holes of a cell culture plate of the sample bearing unit can bear samples; a reagent in the reagent bin can be added into a hole of the cell culture plate by a pipetting arm of the liquid treatment unit, the temperature control unit can control the temperature of the reagent in the hole of the cell culture plate, and the dynamic uniform mixing unit can drive the cell culture plate to reciprocate, so that liquid in the hole of the cell culture plate is uniformly mixed. Reagents are automatically added through the liquid treatment unit, the experiment operation efficiency is improved, pollution caused by manual intervention is avoided, the reagent temperature is controlled through the temperature control unit, and the experiment accuracy is improved; meanwhile, the dynamic uniform mixing unit is used for driving the cell culture plate to reciprocate, so that the reagent incubation uniform mixing effect is enhanced.

Description

Technical Field

[0001] This invention relates to the field of immunocytochemistry technology, and in particular to an immunocytochemical staining instrument and its usage method. Background Technology

[0002] Immunocytochemistry is a cell biology technique based on the specific binding reaction between antigens and antibodies. It is used to qualitatively, locally, and even quantitatively analyze target antigens (such as proteins, peptides, and other biological macromolecules) within cells. Immunocytochemistry requires multiple steps of treatment on cells cultured on coverslips (such as fixation, permeabilization, blocking, and antibody incubation).

[0003] Current immunocytochemistry techniques face numerous challenges in their application: Firstly, automation is lacking. Existing automated equipment (such as Roche's VENTANA) is designed specifically for immunohistochemistry of tissue sections (IHC) and cannot meet the needs of processing cell samples in 24-well plates. Secondly, reagent mixing efficiency is low, and manual shaking operation makes it difficult to ensure uniform reagent coverage of the cell surface within the wells, thus affecting the consistency of staining results. Furthermore, traditional heating modules lack sufficient temperature control precision, failing to achieve independent temperature control for each well, resulting in significant temperature differences between the edge and center wells, affecting the stability of the reaction process. In addition, waste disposal poses significant risks; waste containing toxic reagents (such as Triton X-100 and DAB) can easily cause environmental pollution when manually disposed of. It is worth noting that existing technologies have not yet solved the aforementioned core problems and have obvious limitations. For example, the existing technology disclosed in CN114813924A (DaAn Gene) is a cell staining temperature control device that can only solve temperature control-related problems, but does not address the automation of the entire 24-well plate cell staining process and the issue of reagent mixing. Furthermore, the cell fixation slides designed in the existing technology disclosed in US20180209956A1 (BD) cannot avoid the risk of contamination due to their reliance on open operation. Summary of the Invention

[0004] The purpose of this invention is to provide an immunocytochemical staining instrument and its usage method to solve the problems existing in the above-mentioned related technologies, improve the experimental operation efficiency of immunocytochemical technology, improve the uniformity of reagent mixing, and ensure the stability of the reaction process.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides an immunocytochemical staining instrument, comprising: Workbench; A sample carrying unit, the sample carrying unit including a cell culture plate, wherein the wells of the cell culture plate have coverslips adapted to the wells; A temperature control unit, which can be connected to the cell culture plate and control the temperature of the liquid in the well of the cell culture plate; A liquid processing unit includes a pipette arm and a reagent compartment. The pipette arm is slidably connected to the worktable, and the reagent compartment is capable of holding reagents. The movement of the pipette arm can transfer the reagents in the reagent compartment into the wells of the cell culture plate. A dynamic mixing unit is disposed on the worktable. The dynamic mixing module can be connected to the cell culture plate and drive the cell culture plate to reciprocate so as to make the liquid in the well of the cell culture plate mix evenly.

[0006] Preferably, the temperature control unit includes a temperature control plate located at the bottom of the cell culture plate and abutting against each other, so as to control the liquid temperature in the well of the cell culture plate; The temperature control plate is embedded with multiple sets of Peltiers, and the number and position distribution of the Peltiers are consistent with the number and position distribution of the wells in the cell culture plate.

[0007] Preferably, the liquid processing unit further includes a pipette connected to the pipette arm and communicating with the reagent compartment, and is capable of transferring reagents in the reagent compartment into the wells of the cell culture plate; The number of pipette arms is multiple sets, and the pipette corresponds one-to-one with the pipette arm; The liquid processing unit also includes a waste liquid tank, and the pipette is connected to the waste liquid tank to transfer waste liquid from the wells of the cell culture plate to the waste liquid tank.

[0008] Preferably, the liquid processing unit further includes a pipette, which is slidably disposed on the worktable, a pipette arm is slidably connected to the pipette, and the reciprocating sliding direction of the pipette is perpendicular to the reciprocating sliding direction of the pipette arm, and a pipette is slidably connected to the pipette arm, and the reciprocating sliding direction of the pipette is parallel to the vertical direction; The pipette is connected to the reagent compartment and the waste liquid compartment via a pipetting line.

[0009] Preferably, the dynamic mixing unit includes a positioning frame, a drive ring, a mixing body, and a mixing actuator. The cell culture plate is detachably connected to the positioning frame. When the cell culture plate is connected to the positioning frame, the cell culture plate is located on top of the positioning frame. Both the positioning frame and the drive ring are rotatably connected to the mixing body. The mixing body is connected to the worktable. The mixing actuator is disposed on the mixing body and is driven by the drive ring to drive the drive ring to rotate. The drive ring is driven by the positioning frame, thereby driving the positioning frame and the cell culture plate to rotate, so that the reagents in the wells of the cell culture plate are mixed evenly.

[0010] Preferably, the positioning frame has a positioning block that can abut against the cell culture plate; The positioning frame is also connected to a fixing clip, which is hinged to the positioning frame, and a spring plate is provided between the fixing clip and the positioning frame so that the fixing clip can press the cell culture plate onto the positioning frame.

[0011] Preferably, the drive ring is magnetically connected to the positioning frame; The top of the drive ring has multiple drive magnets arranged circumferentially around the axis of the drive ring, and adjacent drive magnets have opposite polarities. The bottom of the positioning frame has multiple positioning magnets arranged circumferentially around the axis of the drive ring. The positioning magnets and drive magnets are positioned to match each other and there is a gap between them. The mixing driver drives the drive ring to rotate, thereby driving the positioning frame to rotate. Ball bearings are provided between the drive ring, the positioning frame, and the mixing body.

[0012] Preferably, the dynamic mixing unit further includes a drive roller, a drive lever, and a drive connecting rod. The drive roller is rotatably mounted on the mixing body, and the output end of the mixing driver is connected to the drive roller. The drive roller is provided with a plurality of positioning pins. The driving swing arm is rotatably mounted on the mixing body. The first end of the driving swing arm is slidably connected to the driving connecting rod via an elongated hole. The driving connecting rod is connected to the driving ring. The second end of the driving swing arm is connected to a swing block, which can abut against the positioning pin. The rotation axis of the driving swing arm is perpendicular to the rotation axis of the driving roller, and the rotation axis of the driving swing arm is located between the first end and the second end. The rotation of the driving roller can drive the driving swing arm to swing using the positioning pin.

[0013] Preferably, the drive roller has a mounting groove adapted to the positioning pin, the length direction of the mounting groove is parallel to the axis of the drive roller, one end of the positioning pin is slidably disposed in the mounting groove and the relative position of the two can be fixed, and the other end of the positioning pin protrudes out of the mounting groove and can contact the swing block. The swing block is rotatably connected to the drive swing rod, and the contact surface between the swing block and the positioning pin is an arc surface; The mixing body is provided with a mounting column, the driving swing rod is rotatably connected to the mounting column, and a reset element is provided at the rotatable connection between the driving swing rod and the mounting column.

[0014] The present invention also provides a method for using the above-mentioned immunocytochemical staining instrument, comprising the following steps: Cells cultured on coverslips are placed into wells of the cell culture plate; The pipette arm injects the reagent from the reagent compartment into the well of the cell culture plate; the temperature control unit maintains the liquid in the well of the cell culture plate within the experimental temperature range. The dynamic mixing unit is used to move the cell culture plate so that the liquid in the well of the cell culture plate is mixed evenly.

[0015] The present invention achieves the following technical advantages over related technologies: The immunocytochemical staining instrument of the present invention includes a worktable, a sample carrying unit, a temperature control unit, a liquid processing unit, and a dynamic mixing unit. The sample carrying unit includes a cell culture plate, with coverslips adapted to the wells of the cell culture plate. The temperature control unit is connected to the cell culture plate and controls the temperature of the liquid within the wells of the cell culture plate. The liquid processing unit includes a pipette arm and a reagent compartment. The pipette arm is slidably connected to the worktable, and the reagent compartment can hold reagents. Movement of the pipette arm transfers the reagents from the reagent compartment to the wells of the cell culture plate. The dynamic mixing unit is disposed on the worktable, and the dynamic mixing module is connected to the cell culture plate and drives the cell culture plate to reciprocate, thereby ensuring uniform mixing of the liquid within the wells of the cell culture plate.

[0016] The immunocytochemical staining instrument of the present invention features a sample-carrying unit where the coverslips in the wells of the cell culture plate can hold the sample. A liquid handling unit with a pipette arm adds reagents from the reagent compartment to the wells of the cell culture plate. A temperature control unit regulates the temperature of the reagents within the wells. A dynamic mixing unit drives the cell culture plate in reciprocating motion, ensuring uniform mixing of the liquids within the wells. This immunocytochemical staining instrument improves experimental efficiency by using a liquid handling unit to add reagents and avoids contamination caused by manual intervention. The temperature control unit improves experimental accuracy by controlling reagent temperature. Furthermore, the dynamic mixing unit enhances reagent mixing by driving the cell culture plate in reciprocating motion, thus ensuring the stability of the experimental reaction process.

[0017] At the same time, the present invention also provides a method of using the above-mentioned immunocytochemical staining instrument. Naturally, the method of using the immunocytochemical staining instrument of the present invention can also achieve the above-mentioned beneficial effects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or related technologies, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the structure of the immunocytochemical staining instrument disclosed in the embodiments of the present invention; Figure 2 This is a schematic diagram of the dynamic mixing unit of the immunocytochemical staining instrument disclosed in the embodiments of the present invention; Figure 3 This is a partial structural schematic diagram of the dynamic mixing unit of the immunocytochemical staining instrument disclosed in the embodiments of the present invention; Figure 4 This is an isometric view of the positioning frame of the dynamic mixing unit of the immunocytochemical staining instrument disclosed in the embodiments of the present invention; Figure 5 This is an isometric view of the positioning frame of the dynamic mixing unit of the immunocytochemical staining instrument disclosed in the embodiments of the present invention from other angles. Figure 6 This is an isometric schematic diagram of the drive ring of the dynamic mixing unit of the immunocytochemical staining instrument disclosed in the embodiments of the present invention; Figure 7 This is a schematic diagram of the positioning pin of the dynamic mixing unit of the immunocytochemical staining instrument disclosed in the embodiments of the present invention.

[0020] In the diagram: 1. Workbench; 2. Cell culture plate; 3. Pipette arm; 4. Pipette; 5. Pipette holder; 6. Pipette tubing; 7. Positioning frame; 8. Drive ring; 9. Mixing body; 10. Mixing actuator; 11. Positioning block; 12. Fixing clamp; 13. Drive magnet; 14. Positioning magnet; 15. Ball bearing; 16. Drive roller; 17. Drive lever; 18. Drive linkage; 19. Positioning pin; 20. Swing block; 21. Mounting slot; 22. Mounting post. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] The purpose of this invention is to provide an immunocytochemical staining instrument and its usage method to solve the problems existing in the above-mentioned related technologies, improve the experimental operation efficiency of immunocytochemical technology, improve the uniformity of reagent mixing, and ensure the stability of the reaction process.

[0023] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1 This embodiment provides an immunocytochemical staining instrument; please refer to [reference needed]. Figures 1-7 The system includes a workbench 1, a sample carrying unit, a temperature control unit, a liquid handling unit, and a dynamic mixing unit. The sample carrying unit includes a cell culture plate 2, with coverslips fitted to the wells of the cell culture plate 2. The temperature control unit is connected to the cell culture plate 2 and controls the temperature of the liquid in the wells of the cell culture plate 2. The liquid handling unit includes a pipette arm 3 and a reagent compartment. The pipette arm 3 is slidably connected to the workbench 1, and the reagent compartment can hold reagents. The movement of the pipette arm 3 can transfer the reagents in the reagent compartment to the wells of the cell culture plate 2. The dynamic mixing unit is located on the workbench 1 and is connected to the cell culture plate 2, driving the cell culture plate 2 to reciprocate, so that the liquid in the wells of the cell culture plate 2 is mixed evenly.

[0025] The immunocytochemical staining instrument of the present invention features a cover glass slide in the well of the cell culture plate 2 of the sample carrying unit that can hold the sample. The pipette arm 3 of the liquid handling unit adds reagents from the reagent compartment to the well of the cell culture plate 2. The temperature control unit controls the temperature of the reagents in the well of the cell culture plate 2. The dynamic mixing unit drives the cell culture plate 2 to reciprocate, thereby ensuring uniform mixing of the liquids in the well of the cell culture plate 2. This immunocytochemical staining instrument improves experimental efficiency by using the liquid handling unit to add reagents and avoids contamination caused by manual intervention. The temperature control unit improves experimental accuracy by controlling the reagent temperature. Furthermore, the dynamic mixing unit enhances the mixing effect of the cell culture plate 2 by driving its reciprocating motion, which helps ensure the stability of the experimental reaction process.

[0026] It should also be noted that, in practical applications, the immunocytochemical staining instrument of this invention further includes a control unit. The sample carrying unit, temperature control unit, liquid processing unit, and dynamic mixing unit are all communicatively connected to the control unit. The control unit can control the working status of each unit, improving the automation level and efficiency of the immunocytochemical staining instrument. The control unit can be equipped with a touch screen, facilitating researchers to monitor the instrument's working status and perform various control operations. It should be explained that the structure and working principle of the control unit are conventional methods used by those skilled in the art and will not be elaborated upon here.

[0027] In this specific embodiment, the cell culture plate 2 is a 24-well plate. The immunocytochemical staining instrument of the present invention is compatible with 24-well plates to improve the instrument's adaptability and increase experimental efficiency. In practical applications, the cell culture plate 2 can also use other sizes of well plates to adapt to different experimental operations, improving the flexibility and adaptability of the immunocytochemical staining instrument of the present invention. To further improve experimental efficiency, in the specific embodiment of the present invention, the sample carrying unit also includes a well plate identifier to identify the cell culture plates 2 in different experiments, improving the convenience of experimental operation. For example, a high-frequency RFID reader and a QR code scanner are provided, supporting UHF band and QR / DM code recognition. Each cell culture plate 2 is affixed with a unique identifier before loading, and the ID automatically activates various experimental programs built into the immunocytochemical staining instrument (such as "standard ICC procedure" and "rapid staining mode"), realizing "placing and using" automated operation. The well plate identifier is communicatively connected to the control unit and has error alarm and duplicate sample detection functions to prevent operational confusion.

[0028] The temperature control unit includes a temperature control plate located at the bottom of the cell culture plate 2, with the two abutting each other. This temperature control plate controls the liquid temperature within the wells of the cell culture plate 2. Direct contact between the temperature control plate and the cell culture plate 2 improves heat exchange efficiency. To ensure stable contact between the temperature control plate and the cell culture plate 2, an adsorption groove adapted to the cell culture plate 2 can be provided on the temperature control plate. The cell culture plate 2 is placed in the adsorption groove and fixed using magnetic adsorption or other adhesive methods. This ensures reliable contact between the cell culture plate 2 and the temperature control plate, improves the structural stability of both the cell culture plate 2 and the temperature control plate, thereby guaranteeing temperature control and enhancing the reliability of the immunocytochemical staining instrument of this invention. The cell culture plate 2 and the temperature control plate are detachable for convenient subsequent experimental operations.

[0029] In this specific embodiment, multiple sets of Peltiers are embedded within the temperature control plate. The number and positional distribution of the Peltiers correspond to the number and positional distribution of the wells in the cell culture plate 2. The temperature of the liquid within the wells of the cell culture plate 2 is controlled using the Peltiers, achieving bidirectional temperature regulation (heating and cooling) based on the Peltier effect, thus improving the working accuracy of the temperature control unit. Furthermore, the consistency between the number and distribution of the Peltiers and the well positions in the cell culture plate 2 further enhances the temperature control accuracy of each well in the cell culture plate 2.

[0030] To monitor the liquid temperature within the wells of cell culture plate 2, the temperature control unit also includes a temperature sensor. This sensor monitors the reagent temperature in each well of cell culture plate 2, further improving the temperature control accuracy. In practical applications, the temperature control range is 4℃ to 45℃, with an accuracy of ±0.3℃. Simultaneously, a non-contact infrared array sensor can be installed above cell culture plate 2 to collect a full-well temperature distribution map. The temperature data is sent to the control unit, which dynamically adjusts the operating status of each Peltier sensor based on temperature field changes, effectively eliminating temperature differences caused by edge effects and thermal inertia, further improving the reliability of the temperature control unit.

[0031] Specifically, the liquid handling unit also includes a pipette 4, which is connected to a pipetting arm 3 and communicates with a reagent compartment, enabling it to transfer reagents from the compartment to the wells of the cell culture plate 2. The pipetting arm 3 uses the pipette 4 to inject reagents into the wells of the cell culture plate 2. The pipette 4 has a self-sealing tip to ensure reliable reagent dispensing. In this specific embodiment, the self-sealing tip is made of medical-grade polypropylene with a polytetrafluoroethylene coating on the inner wall, significantly reducing droplet residue and protein adsorption. The tip of the self-sealing tip integrates a liquid level sensing electrode, which detects the liquid level in real time through impedance changes, avoiding the aspiration of air bubbles or contact with the bottom of the well. A pressure balancing valve is provided at the top of the self-sealing tip, which automatically adjusts the internal and external pressures during liquid aspiration and dispensing to ensure smooth and accurate liquid transfer.

[0032] In practical applications, multiple sets of pipette arms 3 and reagent compartments can be configured to meet different reagent addition needs. A Peltier filter can also be installed inside the reagent compartment to stabilize the reagent temperature and prevent the inactivation of antibodies and enzymes. A capacitive level sensor can be installed inside the reagent compartment to prompt the user to replenish or replace the reagent when the remaining reagent level falls below a set threshold. The reagent compartment can adopt a split structure for convenient reagent addition; additionally, an airtight seal and an inert gas (such as nitrogen) interface can be provided on the lid of the reagent compartment to further ensure reagent stability. It should be noted that when using inert gas to protect the reagents in the compartment, attention must be paid to pressure balance to ensure smooth reagent discharge. Properly setting the protective gas while ensuring smooth reagent output is a common practice among those skilled in the art and will not be elaborated upon here.

[0033] In this specific embodiment, when there are multiple sets of pipette arms 3, the pipette 4 corresponds one-to-one with the pipette arm 3; the pipette arm 3 drives the pipette 4 to move and complete the reagent dispensing, improving the convenience and efficiency of experimental operation.

[0034] To improve waste liquid treatment efficiency during experiments, the liquid treatment unit also includes a waste liquid chamber. Pipettor 4 is connected to the waste liquid chamber to transfer waste liquid from the wells of cell culture plate 2. After the experiment, pipette 4 is used to aspirate and transport the waste liquid to the waste liquid chamber, achieving waste liquid recycling and preventing contamination. Pipettor 4 is connected to both the reagent chamber and the waste liquid chamber via pipetting tubing 6, ensuring reliable reagent and waste liquid delivery. The pipette tip of pipette 4 can adopt a dual-tube structure, connecting to different waste liquid chambers respectively. This allows for identification of the waste liquid type based on the experiment type and switching of the appropriate tubing during aspiration, improving waste liquid treatment efficiency. Alternatively, a pipette tip with droplet sensing can be selected, automatically stopping aspiration when no liquid flow is detected, preventing excessive drying and cell damage. After waste liquid aspiration, an ethanol-deionized water mixture can be injected into the tubing to rinse residual reagents. The rinsing solution can be discharged through a separate tubing, preventing it from entering the waste liquid chamber and avoiding cross-contamination. The piping material can be fluorinated ethylene propylene copolymer, which has excellent chemical inertness and a smooth inner wall, further reducing the risk of residue.

[0035] In a specific embodiment of the present invention, the liquid processing unit further includes a pipette 5, which is slidably mounted on the worktable 1. A pipette arm 3 is slidably connected to the pipette 5, and the reciprocating sliding direction of the pipette 5 is perpendicular to the reciprocating sliding direction of the pipette arm 3. A pipette 4 is slidably connected to the pipette arm 3, and the reciprocating sliding direction of the pipette 4 is parallel to the vertical direction. The pipette 5 and the pipette arm 3 work together to move and adjust the position of the pipette 4 in the horizontal plane. Furthermore, the pipette 4 can slide vertically relative to the pipette arm 3, thereby adjusting its vertical position. This allows the liquid processing unit to adjust its position in space and inject reagents into the wells of the cell culture plate 2, meeting various experimental needs and improving the reliability of the liquid processing unit. In practical applications, the liquid processing unit can be equipped with a pipetting actuator. The pipetting actuator uses a transmission mechanism to drive the movement of the pipette 5, the pipette arm 3, and the pipette 4, adjusting the reagent injection position and improving the efficiency of the liquid processing unit. Selecting a suitable transmission mechanism to meet power transmission requirements is a common practice among those skilled in the art, and will not be elaborated upon here.

[0036] More specifically, the dynamic mixing unit includes a positioning frame 7, a drive ring 8, a mixing body 9, and a mixing actuator 10. The cell culture plate 2 is detachably connected to the positioning frame 7. When the cell culture plate 2 is connected to the positioning frame 7, the cell culture plate 2 is located on top of the positioning frame 7. Both the positioning frame 7 and the drive ring 8 are rotatably connected to the mixing body 9. The mixing body 9 is connected to the worktable 1. The mixing actuator 10 is disposed on the mixing body 9 and is driven by the drive ring 8 to drive the drive ring 8 to rotate. The drive ring 8 is driven by the positioning frame 7, thereby driving the positioning frame 7 and the cell culture plate 2 to rotate, so that the reagents in the wells of the cell culture plate 2 are mixed evenly. The mixing actuator 10 of the dynamic mixing unit drives the drive ring 8 to rotate, and then the drive ring 8 drives the positioning frame 7 and the cell culture plate 2 to rotate, so that the liquid in the wells of the cell culture plate 2 is mixed evenly, enhancing the reagent mixing effect and improving the mixing operation efficiency.

[0037] In order to fix the position of the cell culture plate 2, the positioning frame 7 has a positioning block 11 that can abut against the cell culture plate 2. The cell culture plate 2 abuts against the positioning block 11 to prevent the cell culture plate 2 from moving and misaligning during movement, which would affect the mixing effect of the reagents.

[0038] Meanwhile, the positioning frame 7 is also connected to a fixing clip 12, which is hinged to the positioning frame 7. A spring plate is also provided between the fixing clip 12 and the positioning frame 7 so that the fixing clip 12 can press the cell culture plate 2 onto the positioning frame 7. Rotating the fixing clip 12 compresses the spring plate, allowing the cell culture plate 2 to extend between the fixing clip 12 and the positioning frame 7. After the cell culture plate 2 is in place, the fixing clip 12 is released. Under the restoring force of the spring plate, one end of the fixing clip 12 presses against the cell culture plate 2, fixing the vertical position of the cell culture plate 2. This further ensures the structural stability of the cell culture plate 2 during the mixing operation. In this specific embodiment, the positioning block 11 can restrict the horizontal displacement of the cell culture plate 2, and the fixing clip 12 can restrict the vertical displacement of the cell culture plate 2. The positioning block 11 and the fixing clip 12 cooperate to fix the position of the cell culture plate 2, ensuring the stability of the cell culture plate 2, and thus ensuring the mixing effect of the reagent in the wells of the cell culture plate 2. In practical applications, in order to improve the uniformity of force on the cell culture plate 2, multiple sets of positioning blocks 11 and fixing clips 12 can be set. The number and distribution of positioning blocks 11 and fixing clips 12 can be adjusted according to the specifications of the cell culture plate 2, so as to improve the flexibility and adaptability of the instrument while ensuring the fixation effect of the cell culture plate 2.

[0039] In this specific embodiment, the drive ring 8 is magnetically connected to the positioning frame 7. The drive ring 8 uses magnetic force to drive the positioning frame 7 to rotate. The magnetic drive is a non-contact transmission method, which has no mechanical wear, smooth movement, and low vibration. This improves the movement stability of the positioning frame 7 and the cell culture plate 2, thereby effectively ensuring the mixing effect of reagents in the wells of the cell culture plate 2 and avoiding cross-contamination.

[0040] Furthermore, the top of the drive ring 8 has multiple drive magnets 13, which are circumferentially arranged around the axis of the drive ring 8, and adjacent drive magnets 13 have opposite polarities. The bottom of the positioning frame 7 has multiple positioning magnets 14, which are circumferentially arranged around the axis of the drive ring 8. The positioning magnets 14 and drive magnets 13 are positioned to match each other, and there is a gap between them. The mixing driver 10 drives the drive ring 8 to rotate, thereby driving the positioning frame 7 to rotate. The drive magnets 13 at the top of the drive ring 8 are arranged in a circumferentially equidistant manner with adjacent magnets having opposite polarities. The positioning magnets 14 at the bottom of the positioning frame 7 are coaxially and equidistantly matched with them, with a gap reserved between them to achieve non-contact engagement. When the mixing driver 10 drives the drive ring 8 to rotate, the magnetic field position of the drive magnets 13 synchronously undergoes circumferential displacement, forming a periodic repulsive-attractive cyclic force with the positioning magnets 14, thereby driving the positioning frame 7 to rotate coaxially with the drive ring 8. The drive ring 8 uses magnetic transmission to drive the positioning frame 7 to rotate. The power transmission is smooth and efficient, and the non-contact transmission eliminates the wear, noise and vibration problems caused by mechanical contact.

[0041] In order to reduce the friction between the drive ring 8 and the positioning frame 7 and the mixing body 9, ball bearings 15 are provided between the drive ring 8 and the positioning frame 7 and the mixing body 9 to convert sliding friction into rolling friction, reduce friction, and further improve the motion stability of the drive ring 8 and the positioning frame 7.

[0042] Meanwhile, the dynamic mixing unit also includes a drive roller 16, a drive swing arm 17, and a drive connecting rod 18. The drive roller 16 is rotatably mounted on the mixing body 9, and the output end of the mixing driver 10 is connected to the drive roller 16. The drive roller 16 is provided with a plurality of positioning pins 19. The drive swing arm 17 is rotatably mounted on the mixing body 9. The first end of the drive swing arm 17 is slidably connected to the drive connecting rod 18 through an elongated hole. The drive connecting rod 18 is connected to the drive ring 8. The second end of the drive swing arm 17 is connected to a swing block 20. The swing block 20 can abut against the positioning pins 19. The rotation axis of the drive swing arm 17 is perpendicular to the rotation axis of the drive roller 16, and the rotation axis of the drive swing arm 17 is located between the first end and the second end. The rotation of the drive roller 16 can push the drive swing arm 17 to swing by means of the positioning pins 19.

[0043] The mixing actuator 10 drives the drive roller 16 to rotate, which in turn drives the positioning pin 19 to rotate. The positioning pin 19 then pushes the drive swing rod 17 to swing, which in turn drives the drive ring 8 to swing back and forth via the drive connecting rod 18. The mixing actuator 10 of this invention utilizes the drive roller 16, drive swing rod 17, and drive connecting rod 18 to drive the drive ring 8 to swing back and forth within a certain angle range, thereby driving the positioning frame 7 and the cell culture plate 2 to swing back and forth, further enhancing the mixing effect of reagents within the wells of the cell culture plate 2.

[0044] In this specific embodiment, the reciprocating swing angle of the drive swing arm 17 is controlled by adjusting the arrangement of multiple positioning pins 19 on the outer peripheral surface of the drive roller 16. The drive roller 16 has mounting grooves 21 adapted to the positioning pins 19. The length direction of the mounting grooves 21 is parallel to the axis of the drive roller 16. One end of the positioning pin 19 is slidably disposed within the mounting groove 21, and their relative position can be fixed. The other end of the positioning pin 19 protrudes from the mounting groove 21 and can contact the swing block 20. The mounting grooves 21 on the outer peripheral surface of the drive roller 16, parallel to its axis, facilitate the installation and position adjustment of the positioning pins 19, thereby satisfying the purpose of adjusting the swing angle range of the drive swing arm 17, improving the motion flexibility of the dynamic mixing unit, and enhancing the adaptability of the dynamic mixing unit. It should also be noted that the mounting grooves 21 can be dovetail grooves to prevent the positioning pins 19 from coming out of the mounting grooves 21, ensuring the structural stability of the positioning pins 19. In practical applications, the positioning pin 19 can be fixed in the mounting groove 21 by a locking nut. Loosening the locking nut can adjust the relative position of the positioning pin 19 and the mounting groove 21. The structure is simple and the adjustment operation is convenient.

[0045] It should also be noted that, in this specific embodiment, the portion of the positioning pin 19 that extends into the mounting groove 21 is a structure that matches the mounting groove 21, the portion of the positioning pin 19 that contacts the swing block 20 is a cylindrical structure, the swing block 20 is rotatably connected to the drive swing rod 17, and the contact surface between the swing block 20 and the positioning pin 19 is an arc surface; this avoids the positioning pin 19 and the swing block 20 from getting stuck and affecting the normal rotation of the drive roller 16, thereby improving the working reliability of the dynamic mixing unit.

[0046] To improve the ease of installation of the drive swing arm 17, a mounting post 22 is provided on the mixing body 9. The middle part of the drive swing arm 17 is rotatably connected to the mounting post 22. One end of the drive swing arm 17 is connected to the swing block 20, and the other end is connected to the drive connecting rod 18. A reset element is provided at the rotatable connection between the drive swing arm 17 and the mounting post 22 to ensure that the drive swing arm 17 can abut against the positioning pin 19, thus ensuring smooth power transmission. The reset element can be a spring or similar device. Under the elastic force of the reset element, the swing block 20 is always in contact with the positioning pin 19, thereby driving the drive swing arm 17 to reciprocate during the rotation of the drive roller 16.

[0047] The immunocytochemical staining instrument of this invention achieves fully enclosed automated staining of cell samples by integrating a 24-well plate holder, high-precision liquid handling, dynamic mixing, zoned temperature control, and waste liquid recovery. The device is compatible with standard consumables, significantly improving experimental efficiency and repeatability, and is suitable for high-throughput drug screening and precise clinical diagnosis.

[0048] Example 2 The present invention also provides a method for using an immunocytochemical staining instrument, which, using the immunocytochemical staining instrument of Example 1, specifically includes the following steps: The cells cultured on the coverslip are placed into the wells of cell culture plate 2; The pipette arm 3 injects the reagent from the reagent compartment into the wells of the cell culture plate 2; the temperature control unit maintains the liquid in the wells of the cell culture plate 2 within the experimental temperature range; The cell culture plate 2 is moved by a dynamic mixing unit to make the liquid in the well of the cell culture plate 2 mix evenly.

[0049] Using the immunocytochemical staining instrument of the present invention, reagents can be conveniently injected into the wells of cell culture plate 2 and the reagent temperature can be controlled. The dynamic mixing unit can drive the cell culture plate 2 to reciprocate and swing, thereby making the liquid in the wells of cell culture plate 2 evenly mixed, enhancing the reagent mixing effect and improving the efficiency of experimental operation.

[0050] Example 3 This embodiment provides a standard HeLa cell immunocytochemical (ICC) staining procedure using the immunocytochemical staining instrument described in Embodiment 1, including the following steps: 1. Sample preparation and loading The user will pre-culture HeLa cells (at a density of 5 × 10⁻⁶) on a round coverslip. 4 Cells / wells are placed in a standard 24-well plate. An RFID tag is attached to the outside of the plate, recording the experiment number "ICC-01" and preset program parameters. The plate is placed in the positioning frame of the sample carrier unit, and the plate reader automatically reads the tag information, and the immunocytochemical staining instrument calls the "standard ICC" program.

[0051] 2. Fixing and Cleaning A pipette was used to draw 200 μL of 4% paraformaldehyde fixative from the refrigerated reagent compartment into each well. The temperature control unit maintained the plate temperature at 4°C, and the reaction was allowed to proceed for 10 minutes. After fixation, waste liquid recovery was initiated, and the waste liquid containing the fixative was aspirated into the hazardous waste compartment. Subsequently, three PBS washes were performed, each with a volume of 300 μL, and the mixture was run for 30 seconds using a dynamic mixing unit to ensure no residue remained in the wells.

[0052] 3. Membrane permeation and sealing The immunocytochemical staining system automatically switched to the permeabilization step, injecting 150 μL of 0.1% Triton X-100 permeabilizer into each well. The temperature control unit raised the temperature to 37°C and incubated for 15 minutes. The dynamic mixing unit used intermittent mode (run for 10 seconds, pause for 20 seconds) to promote uniform mixing of the permeabilizer. After permeabilization, three more PBS washes were performed. Subsequently, 200 μL of 5% BSA blocking buffer was injected into each well, and the dynamic mixing unit ran for 5 minutes to complete the blocking.

[0053] 4. Incubation of primary and secondary antibodies Primary antibody (e.g., anti-α-tubulin mouse monoclonal antibody, diluted 1:500) was diluted and injected into each well in a volume of 150 μL. The temperature control unit was set to 25°C and incubated for 60 minutes. After primary antibody incubation, three PBS washes were performed. Subsequently, HRP-labeled secondary antibody (e.g., goat anti-mouse IgG) was injected, and incubation was performed at 25°C for 30 minutes.

[0054] 5. Color Development and Termination 100 μL of DAB chromogenic substrate was added to each well, and the immunocytochemical staining instrument was activated with an infrared thermal imager to monitor the chromogenic reaction in real time. When the optical density value in the well reached the preset threshold (OD=0.8), the reaction was terminated by injecting stop solution (deionized water), and the waste liquid containing DAB was marked as "hazardous" and aspirated into a dedicated container.

[0055] 6. System self-cleaning and standby After the experiment, the immunocytochemical staining instrument activated its self-cleaning program, flushing the tubing with a 70% ethanol-deionized water mixture, followed by two rinses with deionized water to ensure no residue remained. Afterward, the instrument entered standby mode, ready for the next experiment.

[0056] Example 4 This embodiment provides a rapid immunocytochemical staining procedure using the immunocytochemical staining instrument of Embodiment 1, including the following steps: Temperature control and mixing optimization The temperature control unit raises the incubation temperature of the primary and secondary antibodies to 30°C to accelerate antigen-antibody binding. The dynamic mixing unit operates continuously to improve the reagent diffusion rate.

[0057] Result Validation Compared with the standard procedure in Example 3, the staining intensity and consistency in the rapid mode remained above 90%, meeting the needs of preliminary clinical diagnosis.

[0058] In addition, to ensure the long-term stable operation of the instrument, users can perform the following maintenance procedures periodically: 1. Fluid circuit flushing Perform a "deep cleaning" procedure once a month, using a 1% NaOH solution to flush the fluid path and remove protein residue.

[0059] 2. Temperature calibration The system calibrates the temperature of 24 wells using a standard temperature calibration board (with a built-in NIST-certified PT100 sensor), and automatically records the deviation and corrects the output parameters.

[0060] 3. Mixing uniformity test By injecting methylene blue solution and running the dynamic mixing unit, the color distribution within the well is analyzed using an imaging system to assess the uniformity of mixing. If necessary, the operating parameters of the dynamic mixing unit are adjusted.

[0061] Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this invention. Furthermore, those skilled in the art will recognize that, based on the ideas of this invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. An immunocytochemical staining instrument, characterized in that, include: Workbench; A sample carrying unit, the sample carrying unit including a cell culture plate, wherein the wells of the cell culture plate have coverslips adapted to the wells; A temperature control unit, which can be connected to the cell culture plate and control the temperature of the liquid in the well of the cell culture plate; A liquid processing unit includes a pipette arm and a reagent compartment. The pipette arm is slidably connected to the worktable, and the reagent compartment is capable of holding reagents. The movement of the pipette arm can transfer the reagents in the reagent compartment into the wells of the cell culture plate. A dynamic mixing unit is disposed on the worktable. The dynamic mixing module can be connected to the cell culture plate and drive the cell culture plate to reciprocate so as to make the liquid in the well of the cell culture plate mix evenly.

2. The immunocytochemical staining instrument according to claim 1, characterized in that: The temperature control unit includes a temperature control plate located at the bottom of the cell culture plate and abutting against each other, to control the liquid temperature inside the well of the cell culture plate; The temperature control plate is embedded with multiple sets of Peltiers, and the number and position distribution of the Peltiers are consistent with the number and position distribution of the wells in the cell culture plate.

3. The immunocytochemical staining instrument according to claim 1, characterized in that: The liquid processing unit further includes a pipette, which is connected to the pipette arm and communicates with the reagent compartment, and is capable of transferring the reagent in the reagent compartment into the well of the cell culture plate; The number of pipette arms is multiple sets, and the pipette corresponds one-to-one with the pipette arm; The liquid processing unit also includes a waste liquid tank, and the pipette is connected to the waste liquid tank to transfer waste liquid from the wells of the cell culture plate to the waste liquid tank.

4. The immunocytochemical staining instrument according to claim 3, characterized in that: The liquid processing unit further includes a pipette, which is slidably mounted on the worktable. The pipette arm is slidably connected to the pipette, and the reciprocating sliding direction of the pipette is perpendicular to the reciprocating sliding direction of the pipette arm. The pipette is slidably connected to the pipette arm, and the reciprocating sliding direction of the pipette is parallel to the vertical direction. The pipette is connected to the reagent compartment and the waste liquid compartment via a pipetting line.

5. The immunocytochemical staining instrument according to claim 1, characterized in that: The dynamic mixing unit includes a positioning frame, a drive ring, a mixing body, and a mixing actuator. The cell culture plate is detachably connected to the positioning frame. When the cell culture plate is connected to the positioning frame, the cell culture plate is located on top of the positioning frame. Both the positioning frame and the drive ring are rotatably connected to the mixing body. The mixing body is connected to the worktable. The mixing actuator is disposed on the mixing body and is driven by the drive ring to drive the drive ring to rotate. The drive ring is driven by the positioning frame, thereby driving the positioning frame and the cell culture plate to rotate, so that the reagents in the wells of the cell culture plate are mixed evenly.

6. The immunocytochemical staining instrument according to claim 5, characterized in that: The positioning frame has a positioning block that can abut against the cell culture plate; The positioning frame is also connected to a fixing clip, which is hinged to the positioning frame, and a spring plate is provided between the fixing clip and the positioning frame so that the fixing clip can press the cell culture plate onto the positioning frame.

7. The immunocytochemical staining instrument according to claim 5, characterized in that: The drive ring is magnetically connected to the positioning frame. The top of the drive ring has multiple drive magnets arranged circumferentially around the axis of the drive ring, and adjacent drive magnets have opposite polarities. The bottom of the positioning frame has multiple positioning magnets arranged circumferentially around the axis of the drive ring. The positioning magnets and drive magnets are positioned to match each other and there is a gap between them. The mixing driver drives the drive ring to rotate, thereby driving the positioning frame to rotate. Ball bearings are provided between the drive ring, the positioning frame, and the mixing body.

8. The immunocytochemical staining instrument according to claim 5, characterized in that: The dynamic mixing unit further includes a drive roller, a drive swing arm, and a drive connecting rod. The drive roller is rotatably mounted on the mixing body, and the output end of the mixing driver is connected to the drive roller. The drive roller is provided with a plurality of positioning pins. The driving swing arm is rotatably mounted on the mixing body. The first end of the driving swing arm is slidably connected to the driving connecting rod via an elongated hole. The driving connecting rod is connected to the driving ring. The second end of the driving swing arm is connected to a swing block, which can abut against the positioning pin. The rotation axis of the driving swing arm is perpendicular to the rotation axis of the driving roller, and the rotation axis of the driving swing arm is located between the first end and the second end. The rotation of the driving roller can drive the driving swing arm to swing using the positioning pin.

9. The immunocytochemical staining instrument according to claim 8, characterized in that: The drive roller has a mounting groove that matches the positioning pin. The length direction of the mounting groove is parallel to the axis of the drive roller. One end of the positioning pin is slidably disposed in the mounting groove and the relative position of the two can be fixed. The other end of the positioning pin protrudes from the mounting groove and can contact the swing block. The swing block is rotatably connected to the drive swing rod, and the contact surface between the swing block and the positioning pin is an arc surface; The mixing body is provided with a mounting column, the driving swing rod is rotatably connected to the mounting column, and a reset element is provided at the rotatable connection between the driving swing rod and the mounting column.

10. A method of using the immunocytochemical staining instrument as described in any one of claims 1-9, characterized in that, Includes the following steps: Cells cultured on coverslips are placed into wells of the cell culture plate; The pipette arm injects the reagent from the reagent compartment into the well of the cell culture plate; the temperature control unit maintains the liquid in the well of the cell culture plate within the experimental temperature range. The dynamic mixing unit is used to move the cell culture plate so that the liquid in the well of the cell culture plate is mixed evenly.

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