An in situ RNA sequencer

Abstract

This application relates to the field of fluorescence in situ sequencing technology equipment, and in particular to an in situ RNA sequencer. It includes a housing, a chip adapter, a reagent loading device, and a syringe pump. The chip adapter is detachably mounted on the housing and has a reaction chamber with a reagent channel communicating with the reaction chamber. The reagent loading device includes a reagent loading tray, reagent tube holders, and a multi-way selector valve. Multiple reagent tube holders are provided, and the reagent loading tray has multiple mounting slots spaced apart on its periphery. The reagent tube holders are detachably mounted to the reagent loading tray through these mounting slots. The syringe pump is located within the housing and simultaneously connects the reagent tubes of the reagent tube holders, the multi-way selector valve, and the reagent channel of the chip adapter via tubing, for injecting reagents from the reagent tubes into the reaction chamber of the chip adapter. This application has the advantage of improving the efficiency of sequencing sample preparation.

Description

Technical Field

[0001] This application relates to the field of fluorescence in situ sequencing technology equipment, and in particular to an in situ RNA sequencer. Background Technology

[0002] Spatial transcriptomics can locate and differentiate the active expression of functional genes in specific tissue regions, thus providing important information for basic research and clinical diagnosis. As a groundbreaking new omics research technology, it enables us to detect gene activity in different microenvironments within tissue samples and to map the spatial expression of active genes.

[0003] Current spatial transcriptomics methods are mainly divided into two categories: sequencing-based methods and microscopic imaging-based methods. Microscopic imaging-based methods perform in situ sequencing or multiple rounds of single-molecule fluorescence in situ hybridization and imaging at the original location of RNA in cells or tissues, thereby obtaining a series of signals encoded by different fluorescent colors to detect different genes and directly obtain the spatial location information of the detected genes.

[0004] Based on the aforementioned fluorescence in situ sequencing technology, sequencing sample preparation requires incubation and staining of tissue cell samples. However, current methods typically involve manually adding different reagents directly to a glass slide containing the tissue sample for sequencing sample preparation, resulting in low efficiency in tissue sample preparation. Summary of the Invention

[0005] To improve the efficiency of sequencing sample preparation, this application provides an in situ RNA sequencer.

[0006] The in situ RNA sequencer provided in this application adopts the following technical solution: an in situ RNA sequencer, including a shell, a chip adapter, a reagent mounting device, and a syringe pump; The chip adapter is detachably placed on the housing. The chip adapter is used to mount a glass slide with a tissue sample and form a reaction chamber for incubating the tissue sample. The chip adapter is provided with a reagent channel communicating with the reaction chamber. The reagent mounting device is mounted on the housing. The reagent mounting device includes a reagent mounting plate, a reagent tube holder, and a multi-way selector valve. Multiple reagent tube holders are provided. Multiple mounting slots are spaced apart on the periphery of the reagent mounting plate. The reagent tube holders are detachably mounted to the reagent mounting plate through the mounting slots. The injection pump is housed within the housing. The injection pump connects the reagent tube of the reagent tube holder, the multi-way selector valve, and the reagent channel of the chip adapter through a pipeline, for injecting the reagent in the reagent tube into the reaction chamber of the chip adapter.

[0007] By adopting the above technical solution, a glass slide containing tissue samples is assembled into a chip adapter, and then the chip adapter is placed on a housing and connected to a multi-way selector valve and an injection pump. At the same time, reagent tubes with different reagents are inserted into multiple reagent tube sockets, and then the reagent tube sockets are inserted into a reagent mounting tray through mounting slots. The reagents in the different reagent tubes are injected into the reaction chamber of the chip adapter through the injection pump and the multi-way selector valve to prepare sequencing samples, thereby improving the efficiency of sequencing sample preparation.

[0008] Preferably, the chip adapter includes a channel seat, a sealing film, a cover, and a snap-fit ​​assembly. The reagent channel is disposed on the channel seat and includes a first channel and a second channel. One surface of the channel seat is provided with a first pair of interfaces and a second pair of interfaces. One end of the first channel is connected to the first pair of interfaces, and one end of the second channel is connected to the second pair of interfaces. The other surface of the channel seat is provided with a first sample port and a second sample port. The other end of the first channel is connected to the first sample port, and the other end of the second channel is connected to the second sample port. The sealing film has a cavity opening and is placed on the surface of the channel seat. The first sample port and the second sample port are distributed at both ends of the cavity opening. The first pair of interfaces can be connected to the multi-port selector valve pipeline, and the second pair of interfaces can be connected to the injection pump pipeline. The sealing film, the channel seat, and the glass slide form the reaction chamber. The cover is used to cover the glass slide on the surface of the sealing film, and the snap-fit ​​assembly is connected to the cover and can be snapped onto the channel seat.

[0009] By adopting the above technical solution, a sealing film is first placed on the surface of the channel seat. By adjusting the position of the sealing film, the first sample port and the second sample port are located at opposite ends of the cavity opening. Then, a glass slide is placed on the sealing film to form a reaction cavity. The position of the glass slide is adjusted horizontally so that the tissue sample on the glass slide is located inside the cavity opening. Then, the cover is placed on the glass slide and fixed to the channel seat by a snap-fit ​​assembly. The cover is connected to a multi-port selection valve through the first pair of interfaces and to a syringe pump through the second pair of interfaces. Thus, the reagent in the reagent tube is injected into the reaction cavity for tissue sample incubation and staining. Therefore, contamination during the tissue sample incubation and staining process is reduced, and the accuracy of sequencing sample preparation is improved.

[0010] Preferably, the cover is provided with a transparent viewing window at the position corresponding to the reaction chamber.

[0011] By adopting the above technical solution, the situation of reagents in the reaction chamber can be easily observed through the transparent window, and the tissue sample after the reaction can be directly scanned and imaged through the transparent window.

[0012] Preferably, the surface of the cover facing the channel seat is provided with a limiting groove, the surface of the channel seat is provided with a mating boss, the sealing film is placed on the mating boss, the first sample port and the second sample port are distributed at intervals along the length direction on the mating boss, and the mating boss can be embedded in the limiting groove.

[0013] By adopting the above technical solution, the sealing film and glass slide are limited by the limiting groove on the cover and the mating boss on the channel seat, which facilitates the horizontal adjustment of the sealing film and glass slide, so that the tissue sample is located in the reaction chamber, thus facilitating the installation of the sealing film and glass slide.

[0014] Preferably, the cavity opening includes a connecting portion, a liquid guiding portion, and a reaction portion. Two connecting portions and two liquid guiding portions are provided. The two connecting portions are located at both ends of the cavity opening. The first sample port and the second sample port are respectively located in the two connecting portions. Two liquid guiding portions are provided. The width of the connecting portion is smaller than the width of the reaction portion. The reaction portion is connected to the connecting portion through the liquid guiding portion.

[0015] By adopting the above technical solution, when the reagent enters the reaction chamber through the first sample port, it first passes through the connecting part and then along the liquid guiding part to the reaction part at the position of the tissue sample. Since the width of the connecting part is smaller than the width of the reaction part, the liquid guiding part is inclined towards the reaction part on both sides, so that the reagent diffuses evenly along the liquid guiding part into the entire reaction chamber, thereby uniformly filling the reaction chamber with the reagent, reducing the generation of bubbles in the reaction chamber, and ensuring that the tissue sample reacts fully with the reagent.

[0016] Preferably, two buckle assemblies are provided, and the two buckle assemblies are provided on both sides of the cover; the buckle assembly includes a connecting block and a snap-fit ​​block, one end of the connecting block is rotatably connected to the side wall of the cover, and the other end of the connecting block is rotatably connected to the snap-fit ​​block, and the snap-fit ​​block can snap onto the surface of the channel seat away from the cover.

[0017] By adopting the above technical solution, the connecting blocks on both sides of the cover and the snap-fit ​​blocks facilitate the assembly and disassembly of the cover and the channel seat. At the same time, the snap-fit ​​blocks are fixed on the surface of the channel seat away from the cover, thereby reducing contact with the snap-fit ​​blocks during observation or imaging scanning, thus improving the stability of the cover and the channel seat.

[0018] Preferably, the reagent mounting tray has an adjustment groove facing downwards, the adjustment groove is rotatably connected to a knob cover, the multi-way selector valve is connected to multiple hoses, the groove wall of the adjustment groove is provided with a guide hole corresponding to the mounting groove position, the knob cover is provided with a tube clamping notch on its periphery, and the hoses pass through the tube clamping notch and the guide hole in sequence to extend into the reagent tube of the reagent tube holder.

[0019] By adopting the above technical solution, when it is necessary to replace the reagent, rotate the knob cover to pull the tubing out of the reagent tube, thus facilitating the disassembly and assembly of the reagent tube holder; after replacing the reagent tube, rotate the knob cover back to its original position to insert the tubing into the reagent tube, thus facilitating the installation of the reagent tube holder and the connection between the tubing and the reagent tube.

[0020] Preferably, the device further includes a swaying mechanism, which comprises a swaying platform and a driving component. The swaying platform is rotatably mounted on a base and is used to hold the chip adapter. The swaying platform is provided with inlet and outlet connectors that communicate with a multi-port selector valve and an injection pump. When the chip adapter is placed on the swaying platform, the inlet and outlet connectors can communicate with the reaction chamber of the chip adapter. The driving component is connected to the swaying platform and is used to drive the swaying platform to tilt and intermittently reciprocate the chip adapter in the direction of reagent injection or reagent dispensing.

[0021] By adopting the above technical solution, the chip adapter is placed on the stage, and the chip adapter is connected to the sample inlet and outlet connectors on the stage. When the reagent is injected into the reaction chamber by the syringe pump, the drive unit drives the stage to swing and tilt the chip adapter in the sample injection direction, so that the reagent gradually spreads throughout the reaction chamber, thereby reducing the generation of bubbles in the reaction chamber. After the reagent injection is completed, the drive unit drives the stage to swing the chip adapter back and forth, realizing the dynamic mixing of tissue sample and reagent in the chip adapter, accelerating the full reaction of reagent and tissue sample, and minimizing the incubation time. When the reagent is discharged from the reaction chamber by the syringe pump, the drive unit drives the stage to swing and tilt the chip adapter in the sample outlet direction, thereby accelerating the discharge of reagent and reducing the residue of reagent in the reaction chamber.

[0022] Preferably, the stage base is fixedly provided with a channel side block, a fixed side block, and a limiting side block. The channel side block and the fixed side block are arranged opposite to each other, and the limiting side block is connected between the channel side block and the fixed side block. The channel side block, the fixed side block, and the limiting side block together form a placement groove for placing a chip adapter. The sample inlet / outlet connector is connected to the channel side block, and the side wall of the channel side block is provided with a sample inlet / outlet connection hole. When the chip adapter is located in the placement groove, the sample inlet / outlet connection hole is connected to the chip adapter.

[0023] By adopting the above technical solution, the chip adapter is placed in the placement slot and connected to the chip adapter through the sample inlet / outlet connection hole on the channel side block, thereby realizing the stable entry and exit of reagents while the chip adapter swings.

[0024] Preferably, a clamping assembly is provided on the fixed side block. The clamping assembly includes a clamping block, a spring, and a cam handle. The fixed side block has a through hole through which the clamping block moves. One end of the screw portion of the cam handle is fixedly connected to the fixed side block. The spring is sleeved on the screw portion of the cam handle. One end of the spring abuts against the fixed side block, and the other end abuts against the clamping block. The handle portion of the cam handle abuts against the clamping block.

[0025] By adopting the above technical solution, the chip adapter is placed in the placement slot. By rotating the cam handle, the clamping block abuts against the side wall of the chip adapter, thereby improving the stability of the chip adapter in the placement slot and ensuring stable connection between the chip adapter and the sample inlet / outlet connection hole.

[0026] Preferably, the surface of the swing platform is provided with a heating plate.

[0027] By adopting the above technical solution, the heating plate on the stage conducts heat to the chip adapter, thereby accelerating the incubation of tissue cells and reducing the incubation time.

[0028] Preferably, a magnetic attracting element is embedded on the side wall of the chip adapter, and a magnetic element that attracts the magnetic attracting element is provided on the limiting side block.

[0029] By adopting the above technical solution, the chip adapter is placed on the placement slot and attracted by the magnetic components, thereby pre-positioning and fixing the chip adapter, so as to quickly connect the chip adapter with the sample inlet / outlet docking hole.

[0030] Preferably, it also includes a buffer solution tube and a waste solution tube, and the housing is provided with a receiving groove for inserting and placing the buffer solution tube and the waste solution tube. The buffer solution tube is connected to the multi-way selector valve pipeline; one of the outlet ports of the injection pump is connected to the waste solution pipeline.

[0031] By adopting the above technical solution, the buffer solution tube and the waste liquid tube are inserted into the container, thereby treating the waste liquid in the waste liquid tube and facilitating the addition of buffer solution.

[0032] In summary, this application includes at least one of the following beneficial technical effects: a slide containing tissue samples is assembled into a chip adapter, and then the chip adapter is placed in the placement slot of a rocking device and connected to a multi-way selection valve and an injection pump; reagent tubes with different reagents are inserted into multiple reagent tube holders, and then the reagent tube holders are inserted into a reagent mounting tray through mounting slots; the reagents in different reagent tubes are injected into the reaction chamber of the chip adapter through the injection pump and the multi-way selection valve to prepare sequencing samples, thereby improving the efficiency of sequencing sample preparation. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the overall structure of the in situ RNA sequencer in the embodiments of this application.

[0034] Figure 2 This is a schematic diagram of the exploded structure of the in situ RNA sequencer in the embodiments of this application.

[0035] Figure 3 This is a schematic diagram of the overall structure of the reagent installation device in the embodiments of this application.

[0036] Figure 4 This is an exploded structural diagram of the reagent mounting device and the knob cover in the embodiments of this application.

[0037] Figure 5 This is a schematic diagram of the reagent tube holder in an embodiment of this application.

[0038] Figure 6 This is an exploded structural diagram of the reagent mounting tray and knob cover in an embodiment of this application.

[0039] Figure 7 This is a front view of the chip adapter in an embodiment of this application.

[0040] Figure 8 This is an exploded view of the chip adapter in an embodiment of this application.

[0041] Figure 9 yes Figure 7 Sectional view of AA.

[0042] Figure 10 This is a schematic diagram of the structure of the sealing film in the embodiments of this application.

[0043] Figure 11 This is a schematic diagram of the structure to be installed, including the swing device, chip adapter, and swing platform mounting plate, in the embodiments of this application.

[0044] Figure 12 This is a schematic diagram of the structure of the swing device in the embodiments of this application.

[0045] Figure 13 This is an exploded structural diagram of the fixed side block and the clamping assembly in an embodiment of this application.

[0046] Explanation of reference numerals in the attached drawings: 1. Shell; 11. Base; 12. Receptacle; 13. Buffer solution tube; 14. Waste liquid tube; 15. Liquid level observation port; 16. Slab mounting plate; 161. Bearing seat; 162. Detector; 2. Reagent mounting device; 21. Reagent mounting tray; 211. Mounting slot; 212. Slot; 213. Slot; 214. Adjustment slot; 215. Guide hole; 22. Multi-way selector valve; 23. Reagent tube; 24. Reagent tube holder; 241. Groove; 242. Notched groove; 243. Raised edge; 244. Protrusion; 25. Knob cap; 251. Handle; 252. Rotating locking part; 2521. Tube locking notch; 26. Tube; 3. Chip adapter; 31. Channel seat; 311. First channel; 312. Second channel; 313. First pair of interfaces; 314. Second pair of interfaces; 315. First sample port; 316. Second... 317. Sample port; 318. Mating boss; 32. Magnetic component; 32. Sealing film; 321. Cavity opening; 3211. Connecting part; 3212. Liquid guiding part; 3213. Reaction part; 33. Cover; 331. Transparent window; 332. Limiting groove; 34. Snap-fit ​​assembly; 341. Connecting block; 342. Snap-fit ​​block; 35. Glass slide; 4. Swinging device; 41. Swinging platform; 411. Leaving slope; 412. Trigger element ; 413, heating plate; 42, driving component; 43, sample inlet / outlet connector; 44, channel side block; 441, sample inlet / outlet connection hole; 442, O-ring; 45, fixing side block; 451, through hole; 452, mounting block; 46, limiting side block; 461, magnetic suction component; 47, placement slot; 48, clamping assembly; 481, clamping block; 4811, clearance mounting slot; 482, spring; 483, cam handle; 5, injection pump. Detailed Implementation

[0047] The following is in conjunction with the appendix Figure 1-13 This application will be described in further detail.

[0048] This application discloses an in situ RNA sequencer. (Refer to...) Figure 1 and Figure 2The in situ RNA sequencer includes a housing 1, a reagent mounting device 2, a chip adapter 3, a swaying device 4, and a syringe pump 5; a base 11 is provided at the bottom of the housing 1. The reagent mounting device 2 is mounted on the housing 1 and includes a multi-way selector valve 22 for detachably mounting reagent tubes 23. The multi-way selector valve 22 is connected to the reagent tubes 23 via tubing. The chip adapter 3 has a reaction chamber for incubating tissue samples and a reagent channel connected to the reaction chamber. The swaying device 4 is rotatably connected to the housing 1 and is used to hold the chip adapter 3. The swaying device 4 is connected to the reagent channel of the chip adapter 3 and is used to drive the chip adapter 3 to oscillate back and forth. The syringe pump 5 is located inside the housing 1 and is connected to the multi-way selector valve 22 and the reagent channel of the chip adapter 3 via tubing. It is used to inject the reagent in the reagent tube 23 into the reaction chamber of the chip adapter 3. The housing 1 has two receiving slots 12, and a buffer tube 13 and a waste liquid tube 14 are respectively inserted into the two receiving slots 12. The buffer solution tube 13 is connected to the multi-way selector valve 22; one of the outlet ports of the syringe pump 5 is connected to the waste liquid tube 14. A liquid level observation port 15 communicating with the receiving tank 12 is vertically opened on the side wall of the housing 1.

[0049] Reference Figure 3 and Figure 4 Specifically, the reagent mounting device 2 also includes a reagent mounting tray 21, reagent tube holders 24, and a knob cover 25. Multiple reagent tube holders 24 are provided. Multiple mounting slots 211 are spaced apart on the periphery of the reagent mounting tray 21. The reagent tube holders 24 are detachably mounted to the reagent mounting tray 21 via the mounting slots 211. A multi-way selector valve 22 is fixedly connected to the reagent mounting tray 21, and several connectors of the multi-way selector valve 22 are located in the middle of the reagent mounting tray 21. The multi-way selector valve 22 is located inside the housing 1. Multiple flexible tubes 26 are connected to the connectors of the multi-way selector valve 22, and these flexible tubes 26 extend into the reagent tubes 23 of the reagent tube holders 24. The knob cover 25 is rotatably connected to the reagent mounting tray 21 and is engaged with the flexible tubes 26, used to adjust the insertion or withdrawal of the flexible tubes 26 into or out of the reagent tubes 23. In this embodiment, twelve mounting slots 211 are provided, and each of the twelve mounting slots 211 is equipped with a reagent tube holder 24.

[0050] Reference Figure 5 The reagent tube holder 24 has a groove 241 for inserting and placing the reagent tube 23. The reagent tube 23 has a notch 242 on one side that communicates with the groove 241. The diameter of the groove 241 is slightly smaller than the diameter of the reagent tube 23. The groove 241 with the notch 242 has a certain snap-fit ​​elasticity, which can stably snap the reagent tube 23 onto the reagent tube holder 24 and facilitate the disassembly and assembly of the reagent tube 23.

[0051] Reference Figure 5 and Figure 6 The reagent tube holder 24 has protruding edges 243 on both sides, and slots 212 adapted to the protruding edges 243 are provided on both sides of the mounting slot 211. The slots 212 are inclined inward and downward from the slot opening. One of the slots 212 has a retaining groove 213 on its groove wall, and the protruding edge 243 has a protrusion 244 that mates with the retaining groove 213. During the process of inserting the reagent tube holder 24 into the mounting slot 211, the protruding edge 243 of the reagent tube holder 24 is inserted into the slot 212 at the mounting slot 211, and at the same time, the protrusion 244 on the protruding edge 243 is engaged in the retaining groove 213, thereby improving the stability of the reagent tube holder 24 installed in the mounting slot 211.

[0052] Reference Figure 4 and Figure 6 The reagent mounting tray 21 has an adjustment groove 214 facing downwards. A guide hole 215 is provided on the wall of the adjustment groove 214 corresponding to the mounting slot 211. A flexible tube 26 passes through the guide hole 215 and connects to a multi-way selector valve 22. A knob cover 25 is rotatably connected to the adjustment groove 214. The knob cover 25 includes a handle portion 251 and a rotating locking portion 252. Multiple tube-locking notches 2521 are spaced apart on the periphery of the rotating locking portion 252. The rotating locking portion 252 is rotatably connected within the adjustment groove 214, and the flexible tube 26 passes through the tube-locking notches 2521. When the reagent needs to be replaced, rotating the knob cover 25 pulls the flexible tube 26 out of the reagent tube 23, facilitating the installation and removal of the reagent tube holder 24. After replacing the reagent tube 23, rotating the knob cover 25 back to its original position allows the flexible tube 26 to be inserted into the reagent tube 23, facilitating the installation of the reagent tube holder 24 and the connection between the flexible tube 26 and the reagent tube 23.

[0053] Reference Figure 7 and Figure 8 Specifically, the chip adapter 3 includes a channel seat 31, a sealing film 32, a cover 33, and a snap-fit ​​assembly 34. The reagent channel is set on the channel seat 31, which is rectangular in shape.

[0054] Reference Figure 8 and Figure 9The reagent channel includes a first channel 311 and a second channel 312. One surface of the channel seat 31 is provided with a first pair of interfaces 313 and a second pair of interfaces 314. One end of the first channel 311 is connected to the first pair of interfaces 313, and one end of the second channel 312 is connected to the second pair of interfaces 314. In this embodiment, the first pair of interfaces 313 and the second pair of interfaces 314 are located on one side wall of the channel seat 31. The other surface of the channel seat 31 is provided with a first sample port 315 and a second sample port 316. The other end of the first channel 311 is connected to the first sample port 315, and the other end of the second channel 312 is connected to the second sample port 316. In this embodiment, the first sample port 315 and the second sample port 316 are located on the upper surface of the channel seat 31.

[0055] Reference Figure 8 The sealing sheet 32 ​​has a cavity opening 321 located in the middle of the sealing sheet 32. The sealing sheet 32 ​​is placed on the surface of the channel seat 31. A first sample port 315 and a second sample port 316 are distributed at both ends of the cavity opening 321. A first pair of interfaces 313 can be connected to the pipeline of the multi-way selector valve 22, and a second pair of interfaces 314 can be connected to the pipeline of the syringe pump 5. A glass slide 35 covers the upper surface of the sealing sheet 32. The cavity opening 321 of the sealing sheet 32, the surface of the channel seat 31, and the glass slide 35 form a reaction chamber. In this embodiment, the sealing sheet 32 ​​is made of silicone. The silicone does not react with the reagent and can form a sealed reaction chamber. In addition, it can also act as a buffer to prevent the glass slide 35 from being crushed.

[0056] Reference Figure 8 The cover 33 covers the surface of the glass slide 35, and the snap-fit ​​assembly 34 is connected to the cover 33 and can be snapped onto the channel seat 31. First, the sealing film 32 is placed on the surface of the channel seat 31. By adjusting the position of the sealing film 32, the first sample port 315 and the second sample port 316 are located at the two ends of the cavity port 321. Then, the glass slide 35 is placed on the sealing film 32 to form a reaction cavity. The position of the glass slide 35 is adjusted horizontally so that the tissue sample on the glass slide 35 is located inside the cavity port 321. Then, the cover 33 is placed on the glass slide 35, and the snap-fit ​​assembly 34 fixes the cover 33 to the channel seat 31. The first pair of interfaces 313 are connected to the multi-port selection valve 22, and the second pair of interfaces 314 are connected to the injection pump 5. Thus, the reagent in the reagent tube 23 is injected into the reaction cavity for tissue sample incubation and staining. Therefore, contamination during the tissue sample incubation and staining process is reduced, and the accuracy of sequencing sample preparation is improved.

[0057] Reference Figure 8 Furthermore, in order to facilitate observation of the reagents within the reaction chamber and to enable direct sequencing and sample scanning imaging, a transparent viewing window 331 is provided on the cover 33 at the position corresponding to the cavity opening 321.

[0058] Reference Figure 8 A limiting groove 332 is provided on the surface of the cover 33 facing the channel seat 31. A mating boss 317 is provided on the protrusion 244 of the channel seat 31. The sealing film 32 is placed on the mating boss 317. The first sample port 315 and the second sample port 316 are distributed at intervals along the length direction on the mating boss 317. The mating boss 317 can be embedded in the limiting groove 332. The limiting groove 332 on the cover 33 and the mating boss 317 on the channel seat 31 limit the sealing film 32 and the glass slide 35, thereby facilitating the horizontal adjustment of the sealing film 32 and the glass slide 35, so that the tissue sample is located in the reaction chamber, thus facilitating the installation of the sealing film 32 and the glass slide 35.

[0059] Reference Figure 8 Two latching components 34 are provided, located on both sides of the cover 33. Each latching component 34 includes a connecting block 341 and a latching block 342. One end of the connecting block 341 is rotatably connected to the side wall of the cover 33, and the other end of the connecting block 341 is rotatably connected to the latching block 342. The latching block 342 can latch onto the surface of the channel seat 31 away from the cover 33. The connecting blocks 341 and the latching blocks 342 on both sides of the cover 33 facilitate the assembly and disassembly of the cover 33 and the channel seat 31. At the same time, the latching block 342 is fixed on the surface of the channel seat 31 away from the cover 33, thereby reducing contact with the latching block 342 during observation or imaging scanning, thus improving the stability of the cover 33 and the channel seat 31.

[0060] Reference Figure 10 The cavity opening 321 includes a connecting portion 3211, a liquid guiding portion 3212, and a reaction portion 3213. Two connecting portions 3211 and two liquid guiding portions 3212 are provided. The two connecting portions 3211 are located at opposite ends of the cavity opening 321. A first sample port 315 and a second sample port 316 are located within the two connecting portions 3211, respectively. Two liquid guiding portions 3212 are provided. The width of the connecting portion 3211 is smaller than the width of the reaction portion 3213. The reaction portion 3213 is connected to the connecting portion 3211 through the liquid guiding portion 3212. When the reagent enters the reaction cavity through the first sample port 315, it first passes through the connecting portion 3211 and then along the liquid guiding portion 3212 to the reaction portion 3213. Because the width of the connecting portion 3211 is smaller than the width of the reaction portion 3213, the sides of the liquid guiding portion 3212 are inclined towards the reaction portion 3213, thereby allowing the reagent to diffuse evenly throughout the entire reaction cavity along the liquid guiding portion 3212, ensuring a sufficient reaction in the tissue cells. Furthermore, by adjusting the lengths of the connecting portion 3211 and the reaction portion 3213, the capacity of the reaction chamber can be easily adjusted to accommodate different tissue sample capacities. The capacity of the reaction chamber can also be adjusted by adjusting the thickness of the sealing sheet 32.

[0061] Reference Figure 11 and Figure 12 The swing device 4 includes a swing base 41 and a drive component 42. A swing mounting plate 16 is provided on the housing 1. The swing base 41 is rotatably connected to the swing mounting plate 16. A bearing seat 161 is provided on the swing mounting plate 16. The swing base 41 is rotatably connected to the bearing seat 161 through a bearing. The swing base 41 is used to place the chip adapter 3. A sample inlet / outlet connector 43 is provided on the swing base 41. When the chip adapter 3 is placed on the swing base 41, the sample inlet / outlet connector 43 can communicate with the reaction chamber of the chip adapter 3. The drive component 42 is connected to the swing base 41 and is used to drive the swing base 41 to tilt and intermittently reciprocate the chip adapter 3 in the sample inlet or outlet direction of the reaction chamber. During the injection of reagents into the reaction chamber via the syringe pump 5, the drive unit 42 drives the swing platform 41 to tilt and swing the chip adapter 3 in the injection direction, thereby gradually spreading the reagents throughout the reaction chamber and reducing the generation of bubbles within the chamber. After the reagent injection is complete, the drive unit 42 drives the swing platform 41 to reciprocate, achieving dynamic mixing of the tissue sample and reagents within the chip adapter 3, accelerating the full reaction between the reagents and the tissue sample, and minimizing the incubation time. During the removal of reagents from the reaction chamber via the syringe pump 5, the drive unit 42 drives the swing platform 41 to tilt and swing the chip adapter 3 in the discharge direction, thereby accelerating reagent removal and reducing reagent residue within the reaction chamber. In this embodiment, the drive unit 42 is a motor, which is mounted on the swing platform mounting plate 16. In other embodiments, the drive unit 42 can be a combination of a cylinder and a crank-connecting rod assembly to achieve the reciprocating swing of the swing platform 41.

[0062] Reference Figure 11 and Figure 12 To improve the stability of the chip adapter 3 placement and facilitate communication with the sample inlet / outlet connectors, a platform base 41 is fixedly provided with a channel side block 44, a fixed side block 45, and a limiting side block 46. The channel side block 44 and the fixed side block 45 are disposed opposite each other on the two side walls of the platform base 41. The limiting side block 46 is connected to the side wall of the platform base 41 between the channel side block 44 and the fixed side block 45. The channel side block 44, the fixed side block 45, and the limiting side block 46 together form a placement slot 47 for placing the chip adapter 3. Two sample inlet / outlet connectors are provided, and the two sample inlet / outlet connectors 43 are connected to the channel side block 44. Two sample inlet / outlet connection holes 441 are provided on the side wall of the channel side block 44 facing the fixed side block 45. The sample inlet / outlet connectors communicate with the sample inlet / outlet connection holes 441, with one sample inlet / outlet connector for sample intake and the other for sample output. An O-ring 442 is embedded at the opening of the sample inlet / outlet connection hole 441. With the chip adapter 3 in the placement slot 47, it is connected to the chip adapter 3 through the sample inlet / outlet connection hole 441.

[0063] Reference Figure 11 and Figure 12 The bottom of the swing platform 41 is inclined upwards from the middle to both sides, with a clearance slope 411. A trigger 412 is provided on the clearance slope 411, and a detection element 162 for detecting the trigger 412 is provided on the swing platform mounting plate 16. The detection element 162 controls the operation of the motor through a controller. During the swinging process driven by the motor, the trigger 412 on the clearance slope 411 and the detection element 162 on the swing platform mounting plate 16 control the reciprocating swing angle of the swing platform 41 through the controller. In this embodiment, the detection element 162 is an infrared sensor.

[0064] Reference Figure 12 Two magnetic suction elements 461 are embedded at intervals along the length of the side wall of the limiting side block 46, and magnetic elements 318 that attract each other to the magnetic suction elements 461 are embedded on the opposite two side walls of the channel seat 31. The chip adapter 3 is placed on the placement slot 47 and pre-positioned and fixed by the magnetic elements 318, thereby quickly connecting the chip adapter 3 to the sample inlet / outlet docking hole.

[0065] Reference Figure 12 A heating plate 413 is provided on the surface of the stand 41 located in the placement slot 47. After the reagent is injected, the surface with the transparent window 331 is placed in the placement slot 47. The heating plate 413 conducts heat to the reaction chamber, thereby accelerating the incubation reaction of the tissue sample in the reaction chamber and reducing the time for sequencing sample preparation.

[0066] Reference Figure 12 and Figure 13 To further improve the stability of the chip adapter 3 placed in the placement slot 47 and the stability of its connection with the sample inlet / outlet docking hole, a clamping assembly 48 is provided on the fixed side block 45. The clamping assembly 48 includes a clamping block 481, a spring 482, and a cam handle 483. The fixed side block 45 has a through hole 451, and a mounting block 452 is fixedly installed at the middle position of the through hole 451. The clamping block 481 has a clearance mounting groove 4811 in the middle. The clamping block 481 moves through the through hole 451, and the mounting block 452 is embedded in the clearance mounting groove 4811. One end of the screw part of the cam handle 483 is fixedly connected to the mounting block 452. The spring 482 is sleeved on the screw part of the cam handle 483. One end of the spring 482 abuts against the side wall of the mounting block 452, and the other end abuts against the clearance mounting groove 4811 of the clamping block 481. The handle part 251 of the cam handle 483 abuts against the clamping block 481. The chip adapter 3 is placed in the placement slot 47. By rotating the cam handle 483, the clamping block 481 abuts against the side wall of the chip adapter 3, thereby improving the stability of the chip adapter 3 in the placement slot 47 and making the chip adapter 3 stably dock with the sample inlet / outlet connection hole 441.

[0067] The implementation principle of an in situ RNA sequencer according to an embodiment of this application is as follows: a glass slide 35 containing tissue samples is assembled into a chip adapter 3, and then the chip adapter 3 is placed in the placement slot 47 of the rocking device 4 and connected to the multi-way selection valve 22 and the syringe pump 5; reagent tubes 23 with different reagents are inserted into multiple reagent tube holders 24, and then the reagent tube holders 24 are inserted into the reagent mounting tray 21 through the mounting slot 211; the reagents in the different reagent tubes 23 are injected into the reaction chamber of the chip adapter 3 by the syringe pump 5 and the multi-way selection valve 22 to prepare sequencing samples, thereby improving the efficiency of sequencing sample preparation.

[0068] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. An in situ RNA sequencer, characterized in that: It includes a housing (1), a chip adapter (3), a reagent mounting device (2), and an injection pump (5); The chip adapter (3) is detachably placed on the housing (1). The chip adapter (3) is used to mount a glass slide (35) with a tissue sample and form a reaction chamber for incubating the tissue sample. The chip adapter (3) is provided with a reagent channel communicating with the reaction chamber. The reagent mounting device (2) is mounted on the housing (1). The reagent mounting device (2) includes a reagent mounting plate (21), a reagent tube seat (24), and a multi-way selector valve (22). Multiple reagent tube seats (24) are provided. Multiple mounting slots (211) are spaced apart on the periphery of the reagent mounting plate (21). The reagent tube seat (24) is detachably mounted to the reagent mounting plate (21) through the mounting slots (211). The injection pump (5) is installed inside the housing (1). The injection pump (5) connects the reagent tube (23) of the reagent tube holder (24), the multi-way selector valve (22), and the reagent channel of the chip adapter (3) through the pipeline, so as to inject the reagent in the reagent tube (23) into the reaction chamber of the chip adapter (3). The chip adapter (3) includes a channel seat (31), a sealing film (32), a cover (33), and a snap-fit ​​assembly (34). The reagent channel is disposed on the channel seat (31), and the reagent channel includes a first channel (311) and a second channel (312). One surface of the channel seat (31) is provided with a first pair of interfaces (313) and a second pair of interfaces (314). One end of the first channel (311) is connected to the first pair of interfaces (313), and one end of the second channel (312) is connected to the second pair of interfaces (314). The other surface of the channel seat (31) is provided with a first sample port (315) and a second sample port (316). The other end of the first channel (311) is connected to the first sample port (315), and the other end of the second channel (312) is connected to the first sample port (316). The other end of 312) is connected to the second sample port (316); the sealing soft sheet (32) is provided with a cavity port (321), the sealing soft sheet (32) is placed on the surface of the channel seat (31), the first sample port (315) and the second sample port (316) are distributed at both ends of the cavity port (321), the first pair of interfaces (313) can be connected to the pipeline of the multi-way selector valve (22), and the second pair of interfaces (314) can be connected to the pipeline of the injection pump (5); the sealing soft sheet (32), the channel seat (31) and the glass slide (35) form the reaction chamber; the cover (33) is used to cover the glass slide (35) on the surface of the sealing soft sheet (32), and the snap fastener assembly (34) is connected to the cover (33) and can be snapped onto the channel seat (31).

2. The in situ RNA sequencer according to claim 1, characterized in that: The cover (33) is provided with a transparent window (331) at the position corresponding to the reaction chamber.

3. The in situ RNA sequencer according to claim 1, characterized in that: The cover (33) has a limiting groove (332) on its surface facing the channel seat (31). The channel seat (31) has a protrusion (244) with a mating boss (317). The sealing film (32) is placed on the mating boss (317). The first sample port (315) and the second sample port (316) are distributed at intervals along the length direction on the mating boss (317). The mating boss (317) can be embedded in the limiting groove (332).

4. The in situ RNA sequencer according to claim 1, characterized in that: The cavity opening (321) includes a connecting part (3211), a liquid guiding part (3212), and a reaction part (3213). There are two connecting parts (3211) and two liquid guiding parts (3212). The two connecting parts (3211) are located at both ends of the cavity opening (321). The first sample port (315) and the second sample port (316) are located in the two connecting parts (3211) respectively. There are two liquid guiding parts (3212). The width of the connecting part (3211) is smaller than the width of the reaction part (3213). The reaction part (3213) is connected to the connecting part (3211) through the liquid guiding part (3212).

5. The in situ RNA sequencer according to claim 1, characterized in that: Two buckle assemblies (34) are provided, and the two buckle assemblies (34) are provided on both sides of the cover (33); the buckle assembly (34) includes a connecting block (341) and a snap-fit ​​block (342). One end of the connecting block (341) is rotatably connected to the side wall of the cover (33), and the other end of the connecting block (341) is rotatably connected to the snap-fit ​​block (342). The snap-fit ​​block (342) can snap onto the surface of the channel seat (31) away from the cover (33).

6. The in situ RNA sequencer according to claim 1, characterized in that: The reagent mounting tray (21) has an adjustment groove (214) facing downwards. The adjustment groove (214) is rotatably connected to a knob cover (25). The multi-way selector valve (22) is connected to multiple hoses (26). The groove wall of the adjustment groove (214) is provided with a guide hole (215) corresponding to the mounting slot (211). The knob cover (25) is provided with a tube clamping notch (2521) on its periphery. The hose (26) passes through the tube clamping notch (2521) and the guide hole (215) in sequence and extends into the reagent tube (23) of the reagent tube holder (24).

7. The in situ RNA sequencer according to claim 1, characterized in that: It also includes a swing device (4), which includes a swing base (41) and a drive (42). The swing base (41) is rotatably mounted on a swing base body. The swing base (41) is used to place the chip adapter (3). The swing base (41) is provided with an inlet / outlet connector (43) that communicates with a multi-way selector valve (22) and an injection pump (5). When the chip adapter (3) is placed on the swing base (41), the inlet / outlet connector (43) can communicate with the reaction chamber of the chip adapter (3). The drive (42) is connected to the swing base (41) and is used to drive the swing base (41) to drive the chip adapter (3) to tilt and swing intermittently in the direction of reagent injection or reagent discharge.

8. The in situ RNA sequencer according to claim 7, characterized in that: The stage base (41) is fixedly provided with a channel side block (44), a fixed side block (45), and a limiting side block (46). The channel side block (44) and the fixed side block (45) are arranged opposite to each other. The limiting side block (46) is connected between the channel side block (44) and the fixed side block (45). The channel side block (44), the fixed side block (45), and the limiting side block (46) together form a placement groove (47) for placing the chip adapter (3). The sample inlet / outlet connector (43) is connected to the channel side block (44). The side wall of the channel side block (44) is provided with a sample inlet / outlet connection hole (441). When the chip adapter (3) is located in the placement groove (47), the sample inlet / outlet connection hole (441) is connected to the chip adapter (3).

9. The in situ RNA sequencer according to claim 8, characterized in that: A clamping assembly (48) is provided on the fixed side block (45). The clamping assembly (48) includes a clamping block (481), a spring (482), and a cam handle (483). The fixed side block (45) has a through hole (451). The clamping block (481) moves through the through hole (451). One end of the screw part of the cam handle (483) is fixedly connected to the fixed side block (45). The spring (482) is sleeved on the screw part of the cam handle (483). One end of the spring (482) abuts against the fixed side block (45), and the other end abuts against the clamping block (481). The handle part (251) of the cam handle (483) abuts against the clamping block (481).

10. The in situ RNA sequencer according to claim 7, characterized in that: A heating plate (413) is provided on the surface of the swing stand (41).

11. The in situ RNA sequencer according to claim 8, characterized in that: A magnetic attractor (461) is embedded on the side wall of the chip adapter (3), and a magnetic component (318) is provided on the limiting side block (46) to attract the magnetic attractor (461).

12. The in situ RNA sequencer according to claim 1, characterized in that: It also includes a buffer tube (13) and a waste liquid tube (14). The housing (1) is provided with a receiving groove (12) for inserting the buffer tube (13) and the waste liquid tube (14). The buffer tube (13) is connected to the multi-way selector valve (22). One of the outlet ports of the injection pump (5) is connected to the waste liquid tube (14).

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