Sample treatment system and biological sample treatment method

Abstract

Provided are a sample treatment system and a biological sample treatment method. The sample treatment system comprises a tissue sample carrier and a slide carrier, the tissue sample carrier being used for supporting a biological tissue sample to be treated, and the slide carrier being used for carrying a slide to be tested. The sample treatment system further comprises: a sectioning device, which is movably arranged with respect to the tissue sample carrier and is used for sectioning a biological tissue sample to form a tissue section, the slide to be tested comprising a capture slide that is positioned with respect to the tissue sample carrier to receive a tissue section; a liquid adding device, the liquid adding device being movably arranged with respect to the tissue sample carrier and used for spraying a reagent layer with a predetermined thickness onto the capture slide, the liquid adding device at least comprising a first reagent assembly, and the first reagent assembly comprising a staining reagent and / or a permeabilizing reagent for treating a tissue section; and an optical module, the optical module being capable of performing image acquisition on the slide to be tested. The provided sample treatment system has improved degree of system integration and improved system efficiency.

Description

Sample processing system and biological sample processing method TECHNICAL FIELD

[0001] The present application relates to the technical field of transcriptome sample preparation, in particular to a sample processing system and a biological sample processing method. BACKGROUND

[0002] The spatiotemporal transcriptome (or spatial transcriptome) aims to determine the transcriptome of cells at a specific location / time in a tissue to further discover the role of key cells / rare cells in development, pathology and physiology. The library preparation of the spatiotemporal transcriptome is quite complex, and the core mechanism thereof is to use a capture slide containing spatial coordinate information to capture mRNAs at different positions of a tissue section in situ, and the captured mRNAs contain spatial information of cells in the tissue, so as to achieve the purpose of identifying the spatial position of the transcriptome.

[0003] In the related art, the existing spatiotemporal transcriptome library preparation involves many instruments, including a sample adding device, a sectioning machine, a sequencer (capture slide sequencing), a microscope, a pipetting workstation, etc., resulting in a relatively complex system for preparing a transcriptome sample with spatial information. Moreover, the hardware functions of some instruments are overlapped, which affects the convenience of operation, reduces the working efficiency of the system, and there is room for improvement.

[0004] SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, one object of the present application is to provide a sample processing system, which has high integration and high working efficiency.

[0006] The present application also provides a biological sample processing method.

[0007] According to the sample processing system of the first aspect of the present application, the sample processing system comprises a tissue sample stage and a slide stage, the tissue sample stage is used to support a biological tissue sample to be processed, and the slide stage is used to carry a detection slide. The sample processing system further comprises a sectioning device movably arranged relative to the tissue sample stage and used to cut the biological tissue sample to form a tissue section; the detection slide comprises a capture slide positioned relative to the tissue sample stage to receive the tissue section; a liquid adding device movably arranged relative to the capture slide and used to spray a reagent layer with a predetermined thickness on the capture slide, the liquid adding device at least comprising a first reagent assembly, the first reagent assembly comprising a staining reagent and / or a permeabilization reagent for processing the tissue section; and an optical module used to perform image acquisition on the detection slide.

[0008] According to the sample processing system of the embodiments of the present application, a plurality of devices are arranged in a system, and the plurality of devices process the to-be-detected slides respectively, so that the preparation of the spatiotemporal transcriptome sample can be realized, and the plurality of devices with different functions can be integrated to simplify the sample processing system, prevent the plurality of devices from overlapping in function, reduce the cost, shorten the travel distance of the slides between the plurality of devices, facilitate the operation, and improve the working efficiency of the sample processing system.

[0009] According to some embodiments of the present application, the sample processing system further comprises a blowing device, which at least comprises a first air knife for providing airflow to press the tissue slice against the capture slide.

[0010] In some examples, one side of the tissue sample stage has a mounting position for mounting and fixing the biological tissue sample; when the slide stage moves to the mounting position, the slicing device can cut the biological tissue sample so that the cut tissue slice falls onto the slide stage.

[0011] In some examples, a plurality of slide stages are movably arranged relative to the tissue sample stage. The plurality of slide stages can be sequentially moved to the mounting position to respectively receive a plurality of tissue slices continuously cut by the slicing device from the tissue sample. When one of the slide stages receives the tissue slice, the liquid adding device sprays a reagent layer with a predetermined thickness towards the tissue slice on another slide stage.

[0012] In some examples, the blowing device further comprises a second air knife for providing airflow to remove residual reagent on the capture slide.

[0013] In some examples, the blowing device further comprises a third air knife for providing airflow to collect a sample solution.

[0014] According to some embodiments of the present application, the liquid adding device further comprises a second reagent assembly, which comprises a pretreatment reagent for pretreating the capture slide, the pretreatment reagent comprising a capture probe and a positioning sequence, and the second reagent assembly is used for spraying to form array sites on the capture slide, each of the sites comprising the capture probe and a unique positioning sequence.

[0015] According to some embodiments of the present application, the to-be-detected slide comprises a sequencing slide with a nucleic acid sequencing library, and the liquid adding device further comprises a third reagent assembly, which comprises a sequencing reagent for spraying the sequencing reagent towards the sequencing slide.

[0016] In some examples, the optical module comprises at least a first operation mode and a second operation mode, in the first operation mode, the optical module collects microscopic images of the tissue section, in the second operation mode, the optical module collects fluorescent signals of the nucleic acid sequencing library.

[0017] According to some embodiments of the present application, the sample processing system further comprises a thermal cover device, the thermal cover device is movably arranged with the to-be-tested slide to form a reaction cavity with the to-be-tested slide, and a temperature of the reaction cavity is adjusted according to the reagent layer.

[0018] In some examples, the slide stage is movably arranged to drive the to-be-tested slide to move along a movement track, or the sample processing system comprises a transfer device, the transfer device detachably clamps the to-be-tested slide to move along a movement track; the movement track comprises a patch position corresponding to the tissue sample stage, a reagent exchange position corresponding to the reagent adding device, and an optical detection position corresponding to the optical module.

[0019] According to the biological sample processing method of the second aspect of the present application, the method comprises: supporting a biological tissue sample on a tissue sample stage, moving a slicing device relative to the tissue sample stage to cut the biological tissue sample to generate a tissue section; positioning a capture slide relative to the tissue sample stage to receive the tissue section, the capture slide comprising an array of sites, each of the sites comprising a capture probe, the capture probe comprising a capture sequence and a positioning tag, the capture sequence capturing a target nucleic acid; the target nucleic acid being a nucleic acid sample in the tissue section, or the target nucleic acid being a product of a reverse transcription reaction and / or an amplification reaction and / or a transposition reaction on the nucleic acid sample in the tissue section; after the capture probe contacts the target nucleic acid, performing an extension reaction or a ligation reaction to generate a to-be-tested nucleic acid fragment comprising the positioning tag; moving the tissue section relative to a reagent adding device to form a reagent layer of a predetermined thickness on the tissue section under the drive of the capture slide.

[0020] According to some embodiments of the present application, the positioning of the capture slide relative to the tissue sample stage to receive the tissue section comprises: providing an air flow towards the capture slide to the tissue section by using a blowing device, and pressing the tissue section and the capture slide together.

[0021] According to some embodiments of the present application, the positioning of the capture slide relative to the tissue sample stage to receive the tissue section comprises: sequentially positioning at least two capture slides relative to the tissue sample stage to respectively receive different tissue sections. The different tissue sections may, for example, be formed by continuously cutting the biological tissue sample by the slicing device.

[0022] According to some embodiments of the present application, the capturing slide drives the tissue slice to move relative to the liquid adding device to form a predetermined thickness of reagent layer on the tissue slice, comprising: the liquid adding device sprays different reagent layers on the tissue slice according to a preset reaction sequence; repositioning the capturing slide to the hot cover device to form a reaction chamber, and different reagent layers react with the tissue slice in the reaction chamber; and providing air flow to the capturing slide to remove residual reagent layer on the surface of the tissue slice.

[0023] In some examples, the liquid adding device comprises an inkjet chip, a liquid storage structure located on the inkjet chip and in communication with the inkjet chip, and a reagent storage box in communication with the liquid storage structure, and the inkjet chip sprays reagents to form the reagent layer by using piezoelectric inkjet printing technology or thermal bubble inkjet printing technology.

[0024] In some examples, before the capturing slide drives the tissue slice to move relative to the liquid adding device to form a predetermined thickness of reagent layer on the tissue slice, the sample preparation method further comprises: repositioning the capturing slide, aligning the tissue slice pressed with the capturing slide with the optical module, and generating a microscopic image of the tissue slice.

[0025] According to some embodiments of the present application, the biological tissue sample is a fresh frozen tissue slice or a formalin-fixed paraffin-embedded (FFPE) tissue; when the biological tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue, the formalin-fixed paraffin-embedded (FFPE) tissue constitutes the tissue slice, the biological tissue sample is supported on a tissue sample stage, and the capturing slide is positioned relative to the tissue sample stage to receive the formalin-fixed paraffin-embedded (FFPE) tissue without activating the slicing device.

[0026] In some examples, the different reagent layers include a fixing reagent for fixing the tissue slice to the capturing slide.

[0027] In some examples, the different reagent layers include a permeabilization reagent for releasing the target nucleic acid from the tissue slice.

[0028] In some examples, the different reagent layers include a reverse transcription reagent or an amplification reagent or a transposition reagent.

[0029] In some examples, the sample preparation method further comprises: the capturing probe binds to the target nucleic acid; providing air flow to the capturing slide to remove the tissue slice to obtain a slide capturing the target nucleic acid; and using the liquid adding device to spray a cleavage reagent on the slide to obtain a solution of the target nucleic acid fragment with the positioning label.

[0030] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and / or by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0031] The above and / or additional aspects and advantages of the application will become apparent and be well understood from the description of the embodiments, given below, when taken in conjunction with the drawings, wherein:

[0032] Fig. 1 is a schematic diagram of a structure of a sample processing system according to some embodiments of the application;

[0033] Fig. 2 is a schematic diagram of a partial structure of a sample processing system according to some embodiments of the application;

[0034] Fig. 3 is a schematic diagram of a partial structure of a sample processing system according to some other embodiments of the application;

[0035] Fig. 4 is a schematic diagram of a flow of a biological sample processing method according to some embodiments of the application.

[0036] Reference Signs:

[0037] Sample processing system 100, patch site 101, reaction site 102, liquid exchange site 103, optical detection site 104, to-be-detected slide 200, biological tissue sample 300, reagent layer 400, residual reagent layer 500,

[0038] Guide rail 10, tissue sample stage 20, slicing device 30, liquid adding device 40, optical module 50, first air knife 61, second air knife 62, thermal cover device 70. DETAILED DESCRIPTION

[0039] Embodiments of the application are described in detail below with reference to the attached drawing figures, wherein the same or like reference numerals are used throughout the drawings to refer to the same or like components or elements. The embodiments described below are exemplary and are not intended to limit the application, as interpreted by the appended claims.

[0040] In the description of the present application, it needs to be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation of the present application. In addition, the features defined as "first", "second" can be explicitly or implicitly included one or more features. In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0041] In the description of the present application, it needs to be understood that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0042] The sample processing system 100 according to the embodiments of the present application is described below with reference to FIGS. 1-4.

[0043] As shown in FIGS. 1-4, the sample processing system 100 according to the embodiments of the present application includes a tissue sample stage 20, a slide stage, a slicing device 30, a liquid adding device 40 and an optical module 50.

[0044] The tissue sample stage 20 can support a biological tissue sample 300 to be processed, and the slide stage can carry a slide 200 to be detected. For example, the slide stage can use negative pressure adsorption to adsorb at least one slide 200 to be detected. The slide 200 to be detected can be a biochip, and the biochip can be in an unpackaged form, i.e., an open substrate for fixing the analyte to be detected. The slide 200 to be detected can also be a capture slide prepared from a biochip, i.e., a biochip loaded with a macromolecular structure (e.g., a DNB with spatial coordinate ID and capture sequence polyT) for capturing the analyte to be detected.

[0045] The slicing device 30 can be moved relative to the tissue sample stage 20, the slicing device 30 can cut the biological tissue sample 300 to form a tissue slice, the detection slide 200 includes a capture slide, the capture slide can be positioned relative to the tissue sample stage 20 to receive the tissue slice, the liquid adding device 40 can be moved relative to the tissue sample stage 20, the liquid adding device 40 can spray a reagent layer 400 of a predetermined thickness to the capture slide, which can ensure the experimental effect and save the solution and reduce the cost.

[0046] The liquid adding device 40 at least includes a first reagent assembly, the first reagent assembly can include a staining reagent for processing the tissue slice, the first reagent assembly can include a permeabilization reagent for processing the tissue slice, or the first reagent assembly can include the staining reagent and the permeabilization reagent.

[0047] The optical module 50 can perform image acquisition on the detection slide 200, for example, the optical module 50 can perform image acquisition on the capture slide to realize sequencing of the DNB with different spatial coordinates ID and capture sites polyT on the capture slide; or the optical module 50 can perform image acquisition on the tissue slice on the capture slide, which is beneficial to realize library preparation of the spatiotemporal transcriptome after the preparation of the spatiotemporal transcriptome sample is completed.

[0048] Therefore, multiple devices can be matched, on the basis of realizing the preparation of the spatiotemporal transcriptome sample, multiple devices with different functions can be concentrated together to simplify the sample processing system 100, space can be saved, and the active travel of the detection slide 200 between the multiple devices can be shortened, which is convenient for operation, so that the working efficiency of the sample processing system 100 can be improved and the cost can be reduced.

[0049] According to the sample processing system 100 of the embodiment of the present application, multiple devices are arranged in one system, and the multiple devices process the detection slide 200 respectively, which can realize the preparation of the spatiotemporal transcriptome sample, and can realize the integration of multiple devices with different functions to simplify the sample processing system 100, prevent the multiple devices from overlapping in function to reduce the cost, shorten the active travel of the slide between the multiple devices, facilitate operation, and improve the working efficiency of the sample processing system 100.

[0050] As shown in FIG. 1 and FIG. 2, according to some embodiments of the present application, the sample processing system 100 further comprises a blowing device, which at least comprises a first air knife 61, the first air knife 61 can provide an air flow, the air flow is used for blowing, and the tissue slice can be pressed with the capture slide, wherein the air flow can flow through the biological tissue sample 300 when the tissue sample is cut by the cutting device 30, the curling caused by the internal stress of the biological tissue sample 300 can be overcome, so that the cut tissue slice is kept flat, and the success rate of cutting the biological tissue sample 300 can be improved.

[0051] And by keeping the tissue slice flat, the adhesion effect of the tissue slice and the capture slide can be improved, the success rate of the adhesion of the tissue slice and the capture slide can be improved, the tissue slice on the capture slide can be prevented from being wrinkled or the edge from being raised, the tissue slice can be fully contacted with the capture slide, which is beneficial to improve the capture effect of mRNA and improve the preparation effect of the spatiotemporal transcriptome sample.

[0052] In addition, the air flow can be directed towards the capture slide, so that the air flow can flow through the tissue slice when the tissue slice falls on the capture slide, so as to press the tissue slice downward by air pressure, compared with the technical solution of manually pressing the tissue slice to make the tissue slice adhere to the capture slide, the adhesion between the tissue slice and the capture slide can be more uniform, the adhesion effect of the tissue slice and the capture slide can be improved, and the operation is facilitated.

[0053] According to some embodiments of the present application, the blowing device further comprises a gas supply structure in communication with the air knife, the gas supply structure can be a gas compressor, which can provide compressed gas, such as compressed air or compressed nitrogen, the compressed gas enters the air knife through the gas path, so that the air knife can guide the flow direction of the air flow; of course, the gas supply structure can also be a vacuum pump, and the air knife can use the vacuum negative pressure of the vacuum pump to suck away the reagent layer 400 on the surface of the detection slide 200.

[0054] As shown in FIG. 1, in some examples, the tissue sample stage 20 has a mounting position, which can be located on the lower side of the tissue sample stage 20, the biological tissue sample 300 can be mounted on the mounting position, so that the biological tissue sample 300 is inverted, when the slide stage moves to the lower part of the mounting position, the cutting device 30 can cut the biological tissue sample 300, so that the cut tissue slice falls on the slide stage, which is beneficial to the adhesion of the tissue slice and the capture slide, and facilitates the operation.

[0055] It should be understood that the tissue sample stage 20 can also be arranged on the lower side of the slide stage, and the mounting position is upward, and the cut sample slice can be blown upward by the blowing device (such as the first air knife 62) to straighten the slide stage and adhere to the slide stage.

[0056] It should be understood that in some embodiments of the present application, a plurality of slide stages are movably arranged relative to the tissue sample stage, and the plurality of slide stages can be moved to the mounting position in a preset order to receive a plurality of tissue slices successively cut from the tissue sample by the slicing device, wherein when one of the slide stages receives a tissue slice, the liquid adding device sprays a reagent layer of a predetermined thickness towards the tissue slice on another slide stage.

[0057] As shown in FIGS. 1 and 2, in some examples, the air blowing device further includes a second air knife 62 for providing an air flow to remove residual reagent on the capture slide, wherein the air flow is directed towards the capture slide to remove the residual reagent layer 500 after the reaction on the capture slide, that is, in the process of removing the residual reagent layer 500 by the air blowing device, a new reagent layer 400 can be formed on the capture slide, which can achieve liquid exchange of the capture slide, prevent dilution of the reagent layer 400 due to soaking of the tissue slice in a large amount of buffer reagent, facilitate subsequent reactions of the spatiotemporal transcriptome sample preparation, and shorten the reaction time, while facilitating solution saving, cost reduction.

[0058] In some examples, the air blowing device further includes a third air knife for providing an air flow to collect the sample solution, wherein the air flow can be directed towards the capture slide to collect microliter-level sample solution on the capture slide to the collection device by the air flow, which facilitates operation and does not require collection after dilution, and can increase the sample concentration in the sample solution and improve the preparation effect of the spatiotemporal transcriptome sample.

[0059] According to some embodiments of the present application, the liquid adding device 40 further includes a second reagent assembly including a pretreatment reagent, which can pretreat the capture slide, and the pretreatment reagent includes a capture probe and a positioning sequence, and the second reagent assembly can be sprayed on the capture slide to form array sites, each site including a capture probe.

[0060] For example, the capture slide is sprayed with hundreds of millions of regularly arranged single-stranded DNA nanoballs (DNB), which are obtained by rolling circle replication (RCR) using single-stranded circular DNA as a template, and the diameter of the DNB can be 220 nm, and the distance between the center points of two adjacent DNBs is, for example, within 500 nm, and each DNB has a spatial position sequence (i.e., a positioning tag) corresponding to a coordinate position, which can establish a mapping relationship with the position coordinates.

[0061] Further, the capture sequence such as Poly T is printed on the array site by the liquid adding device 40 to obtain a capture probe having a capture sequence and a positioning tag, which can be used for capture of free target nucleic acids.

[0062] According to some embodiments of the present application, the sample processing system 100 of the present application can also be used for nucleic acid sequence detection, the sample slide 200 to be detected comprises a sequencing slide having a nucleic acid sequencing library, and the liquid adding device 40 further comprises a third reagent assembly comprising a sequencing reagent for spraying the sequencing reagent towards the sequencing slide.

[0063] It should be understood that the sample processing system of the present application can comprise at least two liquid adding devices 40 for spraying different reagents towards the sample slide 200 to be detected.

[0064] As shown in FIG. 3, in some embodiments of the present application, the sample processing system simultaneously processes at least two sample slides 200 to be detected, different liquid adding devices 40a-40b are used to simultaneously spray different reagents towards different sample slides 200 to be detected, and the at least two sample slides 200 to be detected can be moved along the preset path by different slide carriages to the corresponding at least two liquid adding devices 40.

[0065] In another embodiment of the present application, a plurality of mounting positions can be provided on the same slide carriage to receive a plurality of sample slides 200 to be detected.

[0066] According to some embodiments of the present application, the liquid adding device 40 comprises an inkjet chip, a liquid storage structure and a reagent storage box, the liquid storage structure is located on the inkjet chip and communicates with the inkjet chip, and the reagent storage box communicates with the liquid storage structure. The inkjet chip can use piezoelectric inkjet printing technology or thermal bubble inkjet printing technology to spray reagents, so that when the slide carriage moves to the lower part of the inkjet chip, the inkjet chip can spray reagents onto the sample slide 200 to be detected, thereby achieving large-area liquid addition to the sample slide 200 to be detected. This can enable the sample processing system 100 to process large-area tissue sections, which is beneficial to improve the adaptability of the tissue sample.

[0067] It can be understood that the inkjet chip can be an integrated MEMS slide composed of a micro-hole array and a thermal bubble control circuit. The micro-hole array has a very high density (the array pitch can be 40 pm), a high printing frequency (the printing frequency can be greater than 10 kHz), and small printing droplets (the capacity of the droplets can be limited to 9-50 pL), so that the liquid adding device 40 has the characteristics of high printing efficiency and good printing uniformity.

[0068] Of course, for liquid addition of a single reagent, the micro-holes can be arranged in a linear manner, and the reagent can automatically flow from the reagent storage box to the liquid storage structure under the action of gravity. In addition, the liquid adding device 40 can use pressurized spraying technology to add liquid to the slide, which uses a slit to extrude the reagent to achieve liquid addition to the slide.

[0069] As shown in FIG. 1, in some examples, the optical module 50 at least includes a first operation mode and a second operation mode, in the first operation mode, the optical module 50 collects a microscopic image of the tissue section, in the second operation mode, the optical module 50 collects a fluorescent signal of the nucleic acid sequencing library.

[0070] As shown in FIG. 1, according to some embodiments of the present application, the sample processing system 100 further comprises a thermal cover device 70, the thermal cover device 70 is movably arranged with the to-be-tested slide 200, and the thermal cover device 70 can form a reaction cavity with the to-be-tested slide 200, and the temperature of the reaction cavity can be adjusted according to the reagent layer 400, wherein the thermal cover device 70 can heat the to-be-tested slide 200, and the thermal cover device 70 can seal the reaction area of the to-be-tested slide 200, prevent the reagents in the reagent layer 400 from evaporating to the outside, and provide good reaction conditions for the to-be-tested slide 200 or the tissue section, which is conducive to improving the preparation effect of the capture slide and the capture effect of mRNA, and can save solution and reduce cost.

[0071] For example, for the case that the to-be-tested slide 200 is sprayed with a permeation reagent, the thermal cover device 70 is used to maintain the tissue section on the to-be-tested slide 200 in a sealed cavity at, for example, 37°C for, for example, 1-20 minutes, and the optimal permeation time can be, for example, 12 minutes, so that after the tissue section is removed from the to-be-tested slide 200, the to-be-tested slide 200 can still present a complete tissue morphology and a good light signal intensity.

[0072] As shown in FIGS. 1 and 2, in some examples, the slide stage is movably arranged, and the slide stage can drive the to-be-tested slide 200 to move along a movement trajectory; or the sample processing system 100 comprises a transfer device, the transfer device detachably clamps the to-be-tested slide 200 to move along the movement trajectory; the movement trajectory comprises a patch position 101, a liquid exchange position 103, and an optical detection position 104, wherein the patch position 101 corresponds to the tissue sample stage 20, the liquid exchange position 103 corresponds to the liquid adding device 40, and the optical detection position 104 corresponds to the optical module 50, and the movement trajectory further comprises a reaction area, and the reaction position 102 corresponds to the thermal cover device 70, that is, by arranging a plurality of positions, and each position corresponds to a corresponding device, so that the plurality of devices can be functionally divided, and the arrangement of the plurality of devices is more reasonable.

[0073] It can be understood that the slicing device 30 and the blowing device can be located on opposite sides (such as the front and rear sides shown in FIG. 1) of the tissue sample stage 20, which can prevent the slicing device 30 and the blowing device from interfering with each other, improve the stability of the operation of the sample processing system 100, and facilitate the sharing of the blowing device by the patching position 101 and the liquid changing position 103, which can improve the integration of multiple devices in the sample processing system 100, further simplify the sample processing system 100, and prevent devices at different positions from overlapping in function, thereby reducing costs.

[0074] Of course, the liquid adding device 40 can be located on one side (such as the front side or the rear side shown in FIG. 1) of the blowing device, for example, the blowing device can be located between the tissue sample stage 20 and the liquid adding device 40; or the blowing device can be located on one side (such as the rear side shown in FIG. 1) of the tissue sample stage 20 and the liquid adding device 40, which can facilitate shortening the active stroke of the detection slide 200 based on the normal operation of the blowing device, and can improve the working efficiency of the sample processing system 100.

[0075] Alternatively, the patching position 101 and the liquid changing position 103 can each have a blowing device, i.e., the blowing device of the patching position 101 can be arranged on the side (such as the rear side shown in FIG. 1) of the tissue sample stage 20 away from the slicing device 30, and the blowing device of the liquid changing position 103 can be arranged on one side (such as the front side or the rear side shown in FIG. 1) of the liquid adding device 40, which can improve the stability of the operation of the blowing device, and can ensure the normal operation of the sample processing system 100 when a blowing device fails, thereby improving the reliability of the operation of the sample processing system 100.

[0076] According to some embodiments of the present application, the heat cover device 70 includes a heating cover and a movable member, and the movable member can drive the heating cover to move, for example, when the slide carrier moves to the position directly below the heating cover, the heating cover can move in the direction from top to bottom, so that the heating cover is closed on the slide carrier.

[0077] The heating cover can heat the detection slide 200, and the heating cover can cooperate with the slide carrier to seal the reaction area of the detection slide 200, so as to prevent the reagent in the reagent layer 400 from evaporating to the outside when the heating cover is heated, thereby providing good reaction conditions for the capture slide or the tissue slice, which can improve the preparation effect of the capture slide and the capture effect of the mRNA, and can save solution and reduce costs; alternatively, the slide carrier can heat the detection slide 200; or the heating cover and the slide carrier can simultaneously heat the detection slide 200, which can improve the heating effect of the detection slide 200.

[0078] It can be understood that the distance between the heating cover and the capture slide can be 500 μm, and on the basis of improving the heating effect on the capture slide, sufficient reaction space can be reserved for the capture slide or the tissue section, thereby providing good reaction conditions for the capture slide or the tissue section, which is beneficial to improve the preparation effect of the capture slide and improve the capture effect of the mRNA and improve the preparation effect of the spatiotemporal transcriptome sample.

[0079] In other examples, the opposite sides (such as the front and back sides shown in FIG. 1) of the heating cover can have openings, and the slide stage can pass through the two openings in turn during the movement, and when the slide stage moves directly below the heating cover, the slide stage can block the openings to seal the reaction area of the capture slide and prevent the reagents in the reagent layer 400 from evaporating to the outside when the heating cover is heated.

[0080] In some examples, during the preparation of the capture slide, the liquid adding device 40 can spray multiple reagents to the capture slide, so that the slide and the reagents can react, and the heating cover can heat the capture slide and the reagents, which is beneficial to the full reaction of the capture slide and the reagents, improves the preparation effect of the capture slide, and can shorten the reaction time and improve the working efficiency of the sample processing system 100.

[0081] During the capture of the mRNA, the liquid adding device 40 can spray multiple reagents to the capture slide, so that the tissue section and the reagents can react, and the capture slide and the reagents can react, and the heating cover can heat the capture slide and the reagents, which is beneficial to the full reaction of the capture slide, the tissue section and the reagents, improves the preparation effect of the spatiotemporal transcriptome sample, and can shorten the reaction time and improve the working efficiency of the sample processing system 100.

[0082] As shown in FIG. 1, according to some embodiments of the present application, the sample processing system 100 further comprises a transfer module, which can carry the slide stage to move the slide stage below the multiple devices (such as the lower part shown in FIG. 1), and when the slide stage moves directly below each device, the device corresponding to the position of the slide stage can process the tissue section on the to-be-detected slide 200 on the slide stage, which is beneficial to realize the preparation of the spatiotemporal transcriptome sample, and beneficial to concentrate multiple devices with different functions together to simplify the sample processing system 100, which can save space, shorten the movement distance of the to-be-detected slide 200 between the multiple devices, facilitate operation, and thereby improve the working efficiency of the sample processing system 100 and reduce the cost.

[0083] As shown in FIG. 1, in some examples, the transfer module includes a guide rail 10 and a sliding member, the sliding member is arranged on the guide rail 10, and the sliding member can slide relative to the guide rail 10, a slide carrier is arranged on the sliding member, a negative pressure adsorption device can be arranged on the slide carrier to adsorb a slide, and a negative pressure adsorption device can be arranged on the sliding member to adsorb the slide carrier; or, a negative pressure adsorption device can be arranged on the sliding member to directly adsorb the slide.

[0084] It can be understood that a plurality of devices can be arranged along the extension direction of the guide rail 10, for example, the guide rail 10 can extend in the front-rear direction, and the plurality of devices are arranged in the front-rear direction; or, the two ends of the guide rail 10 are connected end to end to form a circular ring, and the plurality of devices are arranged in the circumferential direction of the circular ring, which can further integrate the plurality of devices to save space and shorten the active stroke of the slide carrier, thereby improving the working efficiency of the sample processing system 100.

[0085] In some examples, the transfer module includes a mechanical arm and a mounting table, the mounting table can be arranged at one end of the mechanical arm, the slide carrier can be arranged on the mounting table, a negative pressure adsorption device can be arranged on the slide carrier to adsorb the slide, and a negative pressure adsorption device can be arranged on the mounting table to adsorb the slide carrier; or, a negative pressure adsorption device can be arranged on the mounting table to directly adsorb the slide to be detected 200.

[0086] In some examples, the operator can place the slide to be detected 200 on the carrier, the slide carrier is subjected to negative pressure to adsorb the slide, and the movement of the slide carrier can drive the slide to move to the position directly below the liquid adding device 40, the liquid adding device 40 can spray reagent to the slide to be detected 200 to load DNB with spatial coordinates ID and capture sequence polyT on the slide to be detected 200, thereby achieving preparation of the capture slide.

[0087] Then, the liquid adding device 40 can spray reagent to the capture slide, and the movement of the slide carrier can drive the capture slide to move to the position directly below the optical module 50 to sequence the spatial coordinates or other DNA barcodes on the capture slide, and finally, the movement of the slide carrier can drive the capture slide after sequencing to the patch site 101.

[0088] In some examples, after the preparation and sequencing of the capture slide are completed, the operator can install the frozen tissue sample on the mounting site, and the movement of the slide carrier can drive the capture slide to move to the patch site 101, the slicing device 30 can move to cut the tissue slice, and the air blowing device can blow air to the biological tissue sample to make the flat tissue slice directly fall on the capture slide, and then the air blowing device can blow air to the tissue slice to press the tissue slice on the capture slide.

[0089] The slide stage movement can drive the capture slide movement to the reagent changing position 103, the liquid adding device 40 can spray a fixing reagent (e.g., methanol) to the capture slide to fix the tissue section on the capture slide, and the liquid adding device 40 can spray a staining reagent to the capture slide, the staining reagent can be a cell nucleus dye (e.g., DAPI) or an HE dye, etc., the slide stage movement can drive the capture slide movement to the photographing position, and the optical module 50 can image the cells on the tissue section.

[0090] The slide stage movement can drive the capture slide movement to the reagent changing position 103, the liquid adding device 40 can spray a fixing reagent (e.g., methanol) to the capture slide to fix the tissue section on the capture slide, and the liquid adding device 40 can spray a staining reagent to the capture slide, the staining reagent can be a cell nucleus dye (e.g., DAPI) or an HE dye, etc., the slide stage movement can drive the capture slide movement to the photographing position, and the optical module 50 can image the cells on the tissue section.

[0091] As shown in FIG. 4, the present application also provides a biological sample processing method using the sample processing system 100 described in any of the above embodiments.

[0092] The biological sample processing method of the present application comprises: supporting a biological tissue sample 300 on a tissue sample stage 20, moving a sectioning device 30 relative to the tissue sample stage 20 to cut the biological tissue sample 300 to generate a tissue section; positioning a capture slide relative to the tissue sample stage 20 to receive the tissue section, the capture slide comprising an array of sites, each site having a capture probe fixed thereon in advance, the capture probe comprising a capture sequence and a localization tag, the capture sequence capturing a target nucleic acid, the target nucleic acid being a nucleic acid sample in the tissue section, or the target nucleic acid being a product of a reverse transcription reaction and / or an amplification reaction and / or a transposition reaction of the nucleic acid sample in the tissue section.

[0093] It should be understood that the nucleic acid sample in the tissue section can be processed using, for example, “transposase” and “reverse transcriptase” and “nucleic acid polymerase” known in the art, “transposase” and “reverse transcriptase” and “nucleic acid polymerase” refer to protein molecules or protein molecule aggregates responsible for catalyzing specific chemical reactions and biological reactions.

[0094] After the capture probe contacts the target nucleic acid, an extension reaction or a ligation reaction is performed to generate a test nucleic acid fragment comprising the localization tag.

[0095] For example, the capture sequence comprises a structural sequence that selectively hybridizes to and captures the target nucleic acid of the tissue section.

[0096] The capture slide moves the tissue slice relative to the liquid adding device 40 to form a reagent layer 400 of a predetermined thickness on the tissue slice.

[0097] In some embodiments, the capture sequence is an oligonucleotide sequence capable of capturing mRNA of the tissue sample, including a sequence capable of hybridizing to a poly-A tail of the mRNA, such as including a poly-T oligonucleotide sequence.

[0098] Optionally, the poly-T oligonucleotide sequence includes at least 10 (e.g., at least 20) deoxythymidine residues, and the capture sequence has a length greater than 1 bp.

[0099] In some embodiments, the capture sequence has a length of 1-100 bp, such as 10-50 bp, e.g., 10-30 bp.

[0100] By supporting the biological tissue sample 300 on the tissue sample stage 20, fixation of the biological tissue sample 300 can be achieved to facilitate the sectioning device 30 to cut the biological tissue sample 300 to form a tissue slice; by adhering the tissue slice to the capture slide, the capture effect on the mRNA can be improved, and the preparation effect on the to-be-tested nucleic acid containing the positioning tag can be improved; by providing the reagent layer 400 of a predetermined thickness on the capture slide, the experimental effect can be ensured, and the solution can be saved, and the cost can be reduced.

[0101] According to some embodiments of the present application, the capture slide is positioned relative to the tissue sample stage 20 to receive the tissue slice, including: using the air blowing device to provide an air flow to the tissue slice towards the capture slide, and pressing the tissue slice and the capture slide together.

[0102] By providing the air blowing device to blow air to the tissue slice, the adhesion between the tissue slice and the capture slide can be more uniform, the adhesion effect of the tissue slice and the capture slide can be improved, and the operation is facilitated.

[0103] It should be understood that the biological sample processing method of the present application can process multiple capture slides at the same time, for example, at least two capture slides are sequentially positioned relative to the tissue sample stage to respectively receive different tissue slices, and the different tissue slices are formed by the sectioning device to continuously cut the biological tissue sample.

[0104] According to some embodiments of the present application, the capture slide moves the tissue slice relative to the liquid adding device 40 to form a reagent layer 400 of a predetermined thickness on the tissue slice, including: the liquid adding device 40 sprays different reagent layers 400 to the tissue slice according to a predetermined reaction sequence; repositioning the capture slide to the hot cover device 70 to form a reaction cavity, and the different reagent layers 400 react with the tissue slice in the reaction cavity; and providing an air flow to the capture slide to remove the residual reagent layer 400 on the surface of the tissue slice.

[0105] In some examples, the liquid adding device 40 comprises an inkjet chip, a liquid storage structure located on and in communication with the inkjet chip, and a reagent storage box in communication with the liquid storage structure, and the inkjet chip sprays the reagent to form the reagent layer 400 by using a piezoelectric inkjet printing technology or a thermal foaming inkjet printing technology.

[0106] In some examples, before the capture slide drives the tissue slice to move relative to the liquid adding device 40 to form the reagent layer 400 of a predetermined thickness on the tissue slice, the sample preparation method further comprises: repositioning the capture slide, aligning the tissue slice pressed with the capture slide with the optical module 50, and generating a microscopic image of the tissue slice.

[0107] According to some embodiments of the present application, the biological tissue sample 300 is a fresh frozen tissue slice or a formalin-fixed paraffin-embedded (FFPE) tissue or a deep-frozen tissue, for example, the preparation process of the formalin-fixed paraffin-embedded (FFPE) tissue can include: infiltrating and fixing the biological tissue previously cut into a specified thickness slice in formalin, and then embedding the tissue in paraffin, which can specifically include dehydration, alcohol reagent, xylene treatment, wax immersion, embedding, etc.

[0108] Since the FFPE tissue itself constitutes the tissue slice, when the biological sample processing method is applied to the sample processing system 100 of the present application, the slicing device 30 does not need to be started, and the capture slide can be positioned to receive the formalin-fixed paraffin-embedded (FFPE) tissue relative to the tissue sample stage.

[0109] In some examples, the different reagent layers 400 include a fixing reagent for fixing the tissue slice to the capture slide, and the fixing reagent can be methanol.

[0110] In some examples, the different reagent layers 400 include a permeabilization reagent for releasing target nucleic acids from the tissue slice, and after the tissue slice to which the permeabilization reagent is applied is permeabilized at a preset temperature for a set time length in the reaction cavity surrounded by the capture slide and the thermal cover device 70, the mRNA of the tissue slice is successfully captured by the capture probe, and under a suitable permeabilization temperature and permeabilization time length, a clear, uniform signal and a larger brightness of the capture effect can be obtained.

[0111] In some examples, after the capture is completed, a gas knife or the like can be used to provide a gas flow to the capture slide to remove the tissue slice, so as to obtain a slide on which the target nucleic acid is captured; and the liquid adding device 40 is used to spray a cleavage reagent to the slide to obtain a sample solution with a positioning sequence and the target nucleic acid.

[0112] In some examples, the biological sample processing method of the present application further includes a pre-treatment step of the capture slide. The attachment of capture probes (e.g., DNBs) to the surface of the capture slide with the sample processing system 100 can be performed by any suitable method known in the art, such as by nucleic acid hybridization, biotin-streptavidin binding, thiol binding, photo-activated binding, covalent binding, antibody-antigen, physical confinement via hydrogels or other porous polymers, or the like, or any combination thereof.

[0113] Other configurations and operations of the sample processing system 100 according to the embodiments of the present application are known to those of ordinary skill in the art, and are not described in detail herein. In the description of the present application, "a first feature", "a second feature" can include one or more of the features. Among them, the up-down direction, the left-right direction and the front-rear direction are based on the up-down direction, the left-right direction and the front-rear direction shown in the drawings.

[0114] In the description of the present application, unless explicitly specified and limited, a first feature is "on" or "under" a second feature can include that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, the first feature "on", "above" and "over" the second feature includes that the first feature is directly above and obliquely above the second feature, or only means that the first feature is horizontally higher than the second feature.

[0115] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the exemplary description of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0116] Although the embodiments of the present application have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A sample processing system, characterized by, The sample processing system comprises a tissue sample stage for supporting a biological tissue sample to be processed, and a slide stage for carrying a slide to be detected, and further comprises: a slicing device movably arranged relative to the tissue sample stage for slicing the biological tissue sample to form a tissue slice; the slide to be detected comprises a capture slide positioned relative to the tissue sample stage to receive the tissue slice; a liquid adding device movably arranged relative to the capture slide for spraying a reagent layer of a predetermined thickness on the capture slide, the liquid adding device comprising at least a first reagent assembly, the first reagent assembly comprising a staining reagent and / or a permeabilization reagent for processing the tissue slice; an optical module for image acquisition of the slide to be detected.

2. The sample processing system of claim 1, wherein, Further comprising: a blowing device comprising at least a first air knife for providing an air flow to press the tissue slice against the capture slide.

3. The sample processing system of claim 2, wherein, One side of the tissue sample stage has a mounting position for mounting and fixing the biological tissue sample; when the slide stage moves relative to the mounting position, the slicing device can cut the biological tissue sample so that the cut tissue slice falls onto the slide stage.

4. The sample processing system of claim 3, wherein, A plurality of slide stages are movably arranged relative to the tissue sample stage.

5. The sample processing system of claim 2, wherein, The blowing device further comprises a second air knife for providing an air flow to remove residual reagents on the capture slide.

6. The sample processing system of claim 2, wherein, The blowing device further comprises a third air knife for providing an air flow to collect a sample solution.

7. The sample processing system of claim 1, wherein, The liquid adding device further comprises a second reagent assembly, the second reagent assembly comprising a pretreatment reagent for pretreating the capture slide, the pretreatment reagent comprising a capture probe and a unique positioning sequence, the second reagent assembly being used to spray an array site on the capture slide, each site comprising the capture probe and the unique positioning sequence.

8. The sample processing system of claim 1, wherein, The slide to be detected comprises a sequencing slide having a nucleic acid sequencing library, and the liquid adding device further comprises a third reagent assembly comprising a sequencing reagent for spraying the sequencing reagent towards the sequencing slide.

9. The sample processing system of claim 8, wherein, The optical module comprises at least a first operation mode and a second operation mode, in the first operation mode, the optical module acquires a microscopic image of the tissue slice, and in the second operation mode, the optical module acquires a fluorescent signal of the nucleic acid sequencing library.

10. The sample processing system of claim 1, wherein, Further comprising: a thermal cover device movably arranged with the slide to be detected to form a reaction chamber with the slide to be detected, the temperature of the reaction chamber being adjusted according to the reagent layer.

11. The sample processing system of claim 10, wherein, The slide stage is movably arranged to move the slide to be detected along a movement trajectory, or the sample processing system comprises a transfer device for detachably clamping the slide to be detected to move along a movement trajectory. The movement trajectory comprises a patch position corresponding to the tissue sample stage, a reagent exchange position corresponding to the liquid adding device, and an optical detection position corresponding to the optical module.

12. A method of processing a biological sample, characterized by, ​ supporting a biological tissue sample on a tissue sample stage, moving a slicing device relative to the tissue sample stage to cut the biological tissue sample to generate a tissue slice; positioning a capture slide relative to the tissue sample stage to receive the tissue slice, the capture slide comprising an array of sites, each of the sites comprising a capture probe, the capture probe comprising a capture sequence and a localization tag, the capture sequence capturing a target nucleic acid; the target nucleic acid being a nucleic acid sample in the tissue slice, or the target nucleic acid being a product of a reverse transcription reaction and / or an amplification reaction and / or a transposition reaction on a nucleic acid sample in the tissue slice; after the capture probe contacts the target nucleic acid, performing an extension reaction or a ligation reaction to generate a test nucleic acid fragment comprising the localization tag; moving the capture slide with the tissue slice relative to a liquid dispensing device to form a reagent layer of a predetermined thickness on the tissue slice.

13. The biological sample processing method of claim 12, wherein, positioning the capture slide relative to the tissue sample stage to receive the tissue slice comprises: using a blowing device to provide an air flow to the tissue slice towards the capture slide to press the tissue slice against the capture slide.

14. The biological sample processing method of claim 13, wherein, positioning the capture slide relative to the tissue sample stage to receive the tissue slice comprises sequentially positioning at least two of the capture slides relative to the tissue sample stage to respectively receive different tissue slices.

15. The biological sample processing method of claim 14, wherein, the different tissue slices are formed by the slicing device successively cutting the biological tissue sample.

16. The biological sample processing method of claim 12, wherein, moving the capture slide with the tissue slice relative to a liquid dispensing device to form a reagent layer of a predetermined thickness on the tissue slice comprises: the liquid dispensing device sprays different reagent layers towards the tissue slice according to a pre-set reaction sequence; repositioning the capture slide to a thermal cover device to form a reaction chamber, the different reagent layers and the tissue slice reacting in the reaction chamber; and providing an air flow to the capture slide to remove residual reagent layers on the surface of the tissue slice.

17. The biological sample processing method of claim 12, wherein, the liquid dispensing device comprises an inkjet chip, a reservoir structure on the inkjet chip and in communication with the inkjet chip, and a reagent storage box in communication with the reservoir structure, the inkjet chip using a piezoelectric inkjet printing technology or a thermal bubble inkjet printing technology to spray reagents to form the reagent layer.

18. The biological sample processing method of claim 14, wherein, before moving the capture slide with the tissue slice relative to a liquid dispensing device to form a reagent layer of a predetermined thickness on the tissue slice, the sample preparation method further comprises: repositioning the capture slide to align the tissue slice pressed against the capture slide with an optical module to generate a microscopic image of the tissue slice.

19. The biological sample processing method of claim 12, wherein, the biological tissue sample is a fresh frozen tissue slice or a formalin-fixed paraffin-embedded (FFPE) tissue; when the biological tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue, the formalin-fixed paraffin-embedded (FFPE) tissue constitutes the tissue slice, and the capture slide is positioned relative to the tissue sample stage to receive the formalin-fixed paraffin-embedded (FFPE) tissue without activating the slicing device.

20. The biological sample processing method of claim 16, wherein, The different reagent layers include a fixing reagent for fixing the tissue section to the capture slide; preferably, the different reagent layers include a permeabilization reagent for releasing the target nucleic acid from the tissue section.

21. The biological sample processing method of claim 16, wherein, The different reagent layers include a reverse transcription reagent or an amplification reagent or a transposition reagent.

22. The biological sample processing method of claim 20, wherein, The sample preparation method further includes: The capture probe binds to the target nucleic acid; An air flow is provided towards the capture slide to remove the tissue section, to obtain a slide capturing the target nucleic acid; The liquid adding device is used to spray a cleavage reagent towards the slide to obtain a test nucleic acid fragment with the positioning tag solution.

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