Determination system having multiple detection modes, detection method and mode switching method therefor

Abstract

Disclosed in the present disclosure are a determination system having multiple detection modes and a related method. The determination system having multiple detection modes comprises: a sample stage; an optical module used for scanning a sample under test, the optical module at least having a sequencing mode and a fluorescence staining mode; a driver circuit selectively coupled to mode parameters corresponding to an operation mode of the optical module, the mode parameters at least including a first mode control parameter corresponding to the sequencing mode and a second mode control parameter corresponding to the fluorescence staining mode; and a mode switching circuit coupled to the driver circuit to control the driver circuit on the basis of the sample under test: the mode switching circuit associates different mode parameters with the driver circuit on the basis of the type of the sample under test. The present disclosure achieves multi-mode switching of the detection system, and achieves multiple detection functions on a same platform.

Description

Determination system of multiple detection modes, detection method and mode switching method thereof

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to Chinese patent application No. 2024104784632, filed on April 19, 2024, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biological analysis, in particular, to a determination system of multiple detection modes, a detection method and a mode switching method thereof. BACKGROUND

[0004] Microscopy is one of the key technologies in the field of life sciences, and microscopy is used as an important means for observing biological macromolecules in related detection technologies in the field of life sciences. However, the current related microscopy equipment has a single function, and cannot realize complex and multifunctional detection including, for example, sequencing, multiplexed staining imaging, etc. on the same platform, which highly occupies manpower, material resources and increases additional equipment costs, and therefore there is an urgent need for a device that can realize multifunctional detection at a lower cost. SUMMARY

[0005] The present disclosure aims at the drawbacks of the prior art, and aims at the above-mentioned problems, the present disclosure provides a determination system integrating multiple detection modes.

[0006] The first aspect of the present disclosure provides a determination system integrating multiple detection modes, the system comprising:

[0007] a stage for carrying a sample to be detected;

[0008] an optical system selected from at least one or more of the following: a light source, a lens group, an objective lens;

[0009] a detection system for detecting an optical signal;

[0010] a fluid system for providing multiple reagents for a detection reaction;

[0011] a control system for controlling the switching of multiple detection modes, and / or controlling the optical system, the fluid system and the detection system to complete the detection of the sample to be detected according to a predetermined program in a detection mode.

[0012] In one embodiment, the determination system further comprises an analysis system for receiving information from the detection system, analyzing the detection information and outputting a detection result.

[0013] In one embodiment, the detection system is selected from a camera, a photodiode, an optical sensor, and the like.

[0014] In one embodiment, the plurality of detection modes is selected from a nucleic acid sequencing mode, a tissue sample staining mode, a DNA or RNA in-situ detection mode, a cell or microorganism analysis mode, and the like.

[0015] Another aspect of the present disclosure provides an assay system integrating a plurality of detection modes, wherein the assay system comprises:

[0016] one or more stages for carrying the same or different samples to be detected;

[0017] one or more optical systems selected from at least a combination of one or more of a light source, a lens group;

[0018] one or more detection systems for detecting optical signals;

[0019] one or more fluid systems for providing a plurality of reagents for detection reactions;

[0020] one or more control systems for controlling switching of the plurality of detection modes or detection of the plurality of samples to be detected by the plurality of detection modes simultaneously and in parallel.

[0021] In one embodiment, the control system further comprises:

[0022] a driving circuit coupled to the detection system to collect signal data generated by the sample to be detected, the driving circuit being selectively coupled to mode parameters corresponding to operation modes of the detection system, the mode parameters including at least first mode control parameters corresponding to the sequencing mode and second mode control parameters corresponding to the tissue sample staining mode; and

[0023] optionally, a mode switching circuit coupled to the driving circuit to control the driving circuit based on the sample to be detected: when the sample slide is a sequencing chip, the mode switching circuit associates the first set of mode parameters to the driving circuit, and when the sample slide is a tissue sample slide, the mode switching circuit associates the second set of mode control parameters to the driving circuit.

[0024] In one embodiment, the control system further comprises:

[0025] a plurality of independent driving circuits, each of which is coupled to the detection system to collect signal data generated by the sample to be detected, and each of which can be independently selected to be coupled to a mode parameter corresponding to an operation mode of the detection system, the mode parameter including at least a first mode control parameter corresponding to the sequencing mode, a second mode control parameter corresponding to the tissue sample staining mode, a third control parameter corresponding to the DNA or RNA in-situ detection mode, and a fourth control parameter corresponding to the cell or microorganism analysis mode; and

[0026] Optionally, a plurality of mode switching circuits coupled to the driving circuits to control the driving circuits based on the sample to be detected: when the sample slide is a sequencing chip, the mode switching circuit associates a first set of mode parameters to the driving circuit, and when the sample slide is a tissue sample slide, the mode switching circuit associates a second set of mode control parameters to the driving circuit.

[0027] In one embodiment, the optical system further includes a bright field light source and an excitation light source.

[0028] In one embodiment, the sample to be detected includes a nucleic acid sequencing library fixed on the sample slide, a biological tissue sample, a biological liquid sample, a microorganism sample, or a cell sample.

[0029] In one embodiment, the optical system includes an objective lens; in the sequencing mode, the optical system scans the nucleic acid sequencing library through the objective lens and generates sequence information corresponding to the nucleic acid sequencing library; in the tissue sample staining mode, the optical system scans the biological tissue sample through the objective lens and generates an image corresponding to the biological tissue sample.

[0030] In one embodiment, the optical system includes an objective lens; in the DNA or RNA in-situ detection mode, the optical system scans the biological sample through the objective lens and generates a DNA or RNA in-situ image corresponding to the biological sample; in the cell or microorganism analysis mode, the optical system scans the cell or microorganism sample through the objective lens and generates image information corresponding to the cell or microorganism sample.

[0031] In another aspect of the present disclosure, a detection method for multiple detection modes is disclosed, which comprises the following steps:

[0032] fixing a sample to be detected on a stage;

[0033] selecting a detection mode by a control system, including:

[0034] selecting a first detection mode, starting a first fluid system corresponding to the first detection mode, and controlling the sample to be detected to generate a first optical signal through a first detection reaction; and / or selecting a second detection mode, starting a second fluid system corresponding to the second detection mode, and controlling the sample to be detected to generate a second optical signal through a second detection reaction;

[0035] detecting the first optical signal and / or the second optical signal, and transmitting to an analysis system; and

[0036] analyzing the first optical signal and / or the second optical signal to generate detection information and output a detection result.

[0037] In one embodiment, the first detection mode and the second detection mode are the same detection mode, or the first detection mode and the second detection mode are different detection modes.

[0038] In one embodiment, when the first detection mode and the second detection mode are different detection modes, the first detection reaction and the second detection reaction are different detection reactions.

[0039] In one embodiment, the plurality of detection modes are selected from nucleic acid sequence determination mode, tissue sample staining mode, DNA or RNA in situ detection mode, cell or microorganism analysis mode, etc.

[0040] In one embodiment, the plurality of detection modes can be parallel.

[0041] In one embodiment, the detection reaction corresponding to the sequencing mode is a sequencing reaction, wherein the sequencing reaction is selected from synthesis sequencing reaction, ligation sequencing reaction, single molecule sequencing reaction, etc.

[0042] In one embodiment, the detection reaction corresponding to the tissue sample staining mode is selected from affinity reaction, wherein the affinity reaction is selected from antigen-antibody, protein-aptamer, biotin-streptavidin, nucleotide conjugated antibody-fluorescent probe, etc.

[0043] In one embodiment, the detection reaction corresponding to the DNA or RNA in situ detection mode is selected from probe capture reaction, wherein the probe capture reaction is selected from probe conjugated fluorescence, probe ligation, probe amplification, etc.

[0044] In one embodiment, the detection reaction corresponding to the cell or microorganism analysis mode is selected from surface antigen detection, biological marker detection, positive cell detection analysis, cell transcriptome or epigenetic analysis, etc.

[0045] In one embodiment, the control system comprises at least one processor and an interaction unit, the processor and the interaction unit are coupled with the stage and the fluid system,

[0046] inputting a first instruction, the first instruction comprising: initial information, the initial information comprising at least one of a sample type to be detected, a sequencing scheme, a staining scheme, a kit number, and confirmation information;

[0047] controlling the fluid system to perform a first detection reaction according to the initial information;

[0048] generating a first detection result based on the detection reaction;

[0049] controlling the interaction unit to feed back the first detection result.

[0050] In one embodiment, the fixing of the sample to be detected on the stage comprises: the mounting of a plurality of slides to a plurality of stages, comprising:

[0051] mounting a first slide to the first stage;

[0052] mounting a second slide to the second stage;

[0053] Optionally, the first slide and the second slide are fixed with different sample types, or the first slide and the second slide are fixed with the same sample type.

[0054] In one embodiment, an optical system and a detection system are used to collect optical signals, wherein the optical system comprises a first optical link and a second optical link, and the assay method comprises:

[0055] adjusting the relative position between the optical system and the stage so that any one of the first slide and the second slide is aligned with any one of the first optical link and the second optical link.

[0056] Another aspect of the present disclosure discloses a mode switching method of a multi-mode detection assay system, characterized in that the mode switching method comprises:

[0057] identifying the type of sample to be detected;

[0058] determining a mode parameter selected to be coupled to a driving circuit based on the type of sample to be detected;

[0059] When the sample to be detected is a nucleic acid sequencing library, the mode switching circuit associates a first set of mode parameters to the driving circuit, which drives the optical module into a sequencing mode; when the sample to be detected is a biological tissue sample, a microorganism sample, or a cell sample, the mode switching circuit associates a second set of mode parameters to the driving circuit, which drives the optical module into a fluorescence staining mode or a third mode different from the fluorescence staining mode and the sequencing mode.

[0060] In one embodiment, the third mode is a bright field imaging mode. In one embodiment, the optical system comprises an excitation light source and a bright field light source;

[0061] In one embodiment, in the sequencing mode or the fluorescence staining mode, the excitation light source excites the sample to be detected to generate a fluorescence signal;

[0062] In the bright field imaging mode, the bright field light source irradiates the sample to be detected to generate a bright field signal.

[0063] In one embodiment, the assay system of the plurality of detection modes comprises a control module, which is programmable for controlling the liquid channel module to introduce or discharge reagents into or out of the sample chamber, and for controlling the imaging module to image the sample to be detected.

[0064] In some embodiments, the system further comprises an identification circuit coupled to the mode switching circuit, having an interface for identifying the sample slide type and / or the operation mode.

[0065] In some embodiments, the interface comprises a user input port for receiving inputted slide type information and / or the operation mode.

[0066] In some embodiments, the sample slide has an identifiable type identifier; the interface comprises a scanning mechanism for identifying the type of the sample slide according to the type identifier.

[0067] In some embodiments, the sample to be detected comprises a nucleic acid sequencing library, a biological tissue sample, a biological fluid sample, an environmental sample, a microorganism sample, or a cell sample fixed on the sample slide.

[0068] In some embodiments, the optical module comprises an objective lens; in the sequencing mode, the optical module scans the nucleic acid sequencing library through the objective lens and generates sequence information corresponding to the nucleic acid sequencing library; in the fluorescence staining mode, the optical module scans the biological tissue sample through the objective lens and outputs an image corresponding to the biological tissue sample, the microorganism sample, or the cell sample.

[0069] In some embodiments, the optical module comprises an image sensor and an image processing unit operatively connected to the image sensor, the image processing unit is invoked by the second mode control parameter to receive a plurality of images of corresponding FOVs generated by the image sensor and integrate stitching the plurality of images.

[0070] In some embodiments, the optical module comprises a first optical link and a second optical link independently from each other, the first optical link comprises a first objective lens, the second optical link comprises a second objective lens, the first optical link is in the sequencing mode, and the second optical link is in the same or different mode as the first optical link.

[0071] In some embodiments, the optical module further has a third operation mode different from the sequencing mode and the fluorescent staining mode, the optical module further comprises a third optical link, the first optical link, the second optical link and the third optical link are in any one of the sequencing mode, the fluorescent staining mode and the third operation mode at the same time.

[0072] In some embodiments, the sample stage comprises at least two detection sites for receiving a plurality of sample slides.

[0073] In some embodiments, the assay system of multiple detection modes comprises a liquid path system arranged one-to-one corresponding to the detection sites.

[0074] In another aspect, the present disclosure also provides a detection method of an assay system of multiple detection modes, comprising:

[0075] mounting a slide to a sample stage;

[0076] The mode switching circuit determines a mode parameter selected to be coupled to the driving circuit, the mode parameter corresponding to an operation mode of the optical module, at least comprising a first mode control parameter corresponding to the sequencing mode and a second mode control parameter corresponding to the fluorescent staining mode;

[0077] The driving circuit applies the mode parameter determined by the mode switching circuit to the optical module to switch the optical module between at least the sequencing mode and the fluorescent staining mode.

[0078] In some embodiments, before or after mounting a first slide to a first detection site of the sample stage, the detection method further comprises: loading a first sample to be detected to the first slide, the first sample to be detected comprising a nucleic acid sequencing library, a biological tissue sample, a microbial sample or a cell sample.

[0079] In some embodiments, before or after mounting the second slide to the second detection position of the sample stage, the detection method further comprises: loading a second sample to be detected to the second slide, the second sample to be detected comprising a nucleic acid sequencing library, a biological tissue sample, a microorganism sample, or a cell sample.

[0080] In some embodiments, the first sample to be detected is different from the second sample to be detected.

[0081] In some embodiments, the third operation mode is a bright field imaging mode.

[0082] The technical solution of the present disclosure realizes multiple automatic detection functions, has simple operation steps, more effectively utilizes resources, and saves equipment costs. BRIEF DESCRIPTION OF DRAWINGS

[0083] The above and / or additional aspects and advantages of the present disclosure will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:

[0084] Non-limiting and non-exhaustive embodiments of the present disclosure are described with reference to the following drawings, in which:

[0085] FIG. 1 is a simplified schematic diagram of a detection system with multiple detection modes according to an embodiment of the present disclosure;

[0086] FIG. 2 is a schematic diagram of the optical module of the detection system with multiple detection modes according to an embodiment of the present disclosure and the sample stage;

[0087] FIG. 3 is a schematic diagram of the optical module of the detection system with multiple detection modes according to an embodiment of the present disclosure and the controller;

[0088] FIG. 4 is a partial schematic diagram of the optical module of the detection system with multiple detection modes according to an embodiment of the present disclosure;

[0089] FIG. 5A is another schematic diagram of the detection system with multiple detection modes according to an embodiment of the present disclosure;

[0090] FIG. 5B is a schematic diagram of the optical path of the detection system with multiple detection modes according to an embodiment of the present disclosure in a fluorescent staining mode;

[0091] FIG. 5C is a schematic diagram of the detection of the detection system with multiple detection modes according to an embodiment of the present disclosure in a sequencing mode;

[0092] FIG. 6 is a schematic diagram of a detection method of the detection system with multiple detection modes according to an embodiment of the present disclosure;

[0093] FIG. 7 is a schematic diagram of a mode switching method of the detection system with multiple detection modes according to an embodiment of the present disclosure.

[0094] FIG8 shows a schematic diagram of the specific structure of a multifunctional biochemical detection system according to an exemplary embodiment of the present disclosure.

[0095] 9A-9B show schematic diagrams of a sample slide according to an exemplary embodiment of the present disclosure, FIG. 9A shows an assembly diagram of the sample slide, and FIG. 9B shows an exploded view of the assembly of the sample slide.

[0096] Figures 10A-10D are schematic diagrams showing the process of adhering tissue sections to sample slides. Figure 10A shows the process of adhering tissue sections to the slide; Figure 10B shows the state after the tissue sections are adhered to the slide; Figure 10C shows the double-sided tape being adhered to the slide; and Figure 10D shows the sample slide assembly being completed by covering the cover glass with the frame.

[0097] FIG11 shows a schematic diagram of a sample carrier adapted to a fixing fixture. The chip is placed on the chip fixing position of the base, and downward pressure is applied to the pressing block to compress the chip.

[0098] Figure 12 shows a schematic diagram of the liquid circuit module of a measurement system with multiple detection modes according to an exemplary embodiment of the present disclosure, wherein: 1. Reagent tank; 2. Sample slide; 3. Selection valve; 4. Injection pump; 5. Waste liquid barrel.

[0099] FIG13 is a photograph of a mouse brain slice tissue taken by the measurement system with multiple detection modes according to an exemplary embodiment of the present disclosure in a fluorescent staining mode.

[0100] FIG14 is a schematic diagram of the structure of a computer terminal implementing software selectability in an exemplary embodiment of the present disclosure.

[0101] FIG15 is a diagram showing the in situ mRNA detection results obtained using RNA in situ hybridization sequencing technology in one embodiment of the present disclosure.

[0102] FIG16 is a flow chart of a multi-mode detection result analysis module in one embodiment of the present disclosure.

[0103] FIG17 shows the results of detecting single-cell transcriptomes in one embodiment of the present disclosure. DETAILED DESCRIPTION

[0104] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0105] It should be noted that the terms "first", "second", etc. are used only for descriptive purposes, and should not be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present disclosure, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0106] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. For ranges, the endpoints or any other values are approximations that are understood to be variable. For numerical values, the endpoints are understood to be approximate values that are understood to be variable.

[0107] In the present disclosure, the term "comprising" or "including" is an open-ended expression that includes the stated features but not excluding other features.

[0108] In the present disclosure, the terms "longitudinal", "lateral", "upper", "lower", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present disclosure and simplifying the description, and cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present disclosure.

[0109] In the present disclosure, unless otherwise explicitly specified and limited, the terms "connected", "connected", "fixed" and the like should be understood broadly, for example, can be fixedly connected, or can be detachably connected, or can be integrated; can be mechanically connected, or can be electrically connected; can be directly connected, or can be indirectly connected through an intermediate medium; can be the internal communication of two elements or the interaction relationship between two elements, unless otherwise explicitly limited. For those skilled in the art, the specific meaning of the above terms in the present disclosure can be understood according to the specific circumstances.

[0110] In the present disclosure, unless otherwise explicitly specified and limited, the first feature "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature "above", "over" and "on" the second feature can be that the first feature is directly above or obliquely above the second feature, or only indicates that the horizontal height of the first feature is higher than that of the second feature. The first feature "below", "under" and "under" the second feature can be that the first feature is directly below or obliquely below the second feature, or only indicates that the horizontal height of the first feature is less than that of the second feature.

[0111] In this document, unless otherwise explicitly specified and limited, the terms "system", "module", "apparatus", and the like are to be interpreted broadly and have essentially the same definition and meaning, and can be used interchangeably. For example, a fluid system is equivalent to a fluid module, an optical module is equivalent to an optical apparatus, a carrier can be equivalent to a sample stage, a slide can be equivalent to a chip, and the like, all of which are well known to those skilled in the art as similar concepts.

[0112] Omics Analysis refers to the application of a series of high-throughput, large-scale, multi-dimensional techniques to study the collection of various related molecules and their interactions within an organism. The term "Omics" is derived from concepts such as Genome, Proteome, Transcriptome, Metabolome, etc. in biology, each "Omics" represents a complete set of a specific type of molecules within an organism.

[0113] Genome refers to genomic information from a subject, which can be, for example, at least a portion or all of the subject's genetic information. The genome can be encoded in DNA or RNA. The genome can include coding regions that encode proteins as well as non-coding regions. The genome can include the sequences of all chromosomes in an organism. For example, the human genome has a total of 46 chromosomes. The sequences of all of these together can constitute the human genome.

[0114] Cytomics refers to the comprehensive study of all components within a cell, including the structure, function, behavior of the cell, and the interactions between cells. Cytomics covers all levels from a single cell to a population of cells, aiming to reveal the complexity and diversity of cells.

[0115] Transcriptome refers to the study of the collection of RNA molecules, particularly mRNA, which represent the expression of genes at a specific time and under certain conditions. Transcriptome analysis helps to understand how genes are regulated and their roles in biological processes.

[0116] Proteome refers to the comprehensive study of all proteins in an organism, cell, or tissue, including their expression, function, interactions, and dynamic changes. The goal of proteomics is to establish a complete protein catalog, including protein identification, quantification, modification, and functional analysis.

[0117] Metabolome refers to the study of the collection of all small molecule metabolites within an organism. Metabolome analysis can reveal changes in metabolic pathways and the roles of metabolites in biological processes.

[0118] Epigenome, refers to the epigenetic regulatory mechanisms of gene expression, such as DNA methylation, histone modification, etc. These regulatory mechanisms do not change the DNA sequence itself, but can affect the activity of genes.

[0119] Sample, also known as a specimen, refers to biological samples of different origins. Biological samples can be nucleic acid samples, cell samples, microbial samples, or protein samples. Biological samples can be derived from another sample. The sample can be a tissue sample, such as a biopsy, core biopsy, needle aspiration, or fine needle aspiration. The sample can be a fluid sample, such as a blood sample, a urine sample, or a saliva sample. The sample can be a skin sample. The sample can be a cheek swab. The sample can be a plasma or serum sample. The sample can be a cell-free or cell-free sample. The cell-free sample can include extracellular polynucleotides. Extracellular polynucleotides can be isolated from a body sample, which can be selected from blood, plasma, serum, urine, saliva, mucosal discharge, sputum, feces, and tears. It can also be a sample of viruses, bacteria, etc. Different origins refer to the fact that biological samples can be derived from human bodies, animal bodies, plant bodies, microbial communities, environmental samples, and even space samples, etc.

[0120] Slide is a common experimental tool in biology and omics research, used to fix biological samples such as cells, tissue sections or other biological materials on a flat surface for observation, analysis and various biological experiments. It can include slides of different materials, such as glass, silicon-based, resin, high polymer, metal, metal oxide, etc. It can also be divided according to its use, such as sequencing chips, which can include closed or open sequencing chips, semiconductor chips, nanopore chips, microscope slides, tissue microarrays (TMAs), cell arrays, protein arrays, biochips, microarrays, flow cytometry slides, and tissue sections.

[0121] Sequencing, a new type of genetic testing technology, can analyze and determine the full sequence of gene bases from blood or saliva, thereby predicting the possibility of suffering from various diseases, individual behavior characteristics and behavior rationality. Sequencing generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. Polynucleotides can be, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single-stranded DNA). Sequencing can be performed by various systems currently available, such as but not limited to sequencing systems of MGI, Sequencing, Illumina, Pacific Biosciences, Oxford Nanopore or Life Technologies (Ion Torrent). Alternatively, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR or real-time PCR) or isothermal amplification. Such devices can provide multiple raw genetic data corresponding to the genetic information of a subject (e.g., a human), as generated by the device from a sample provided by the subject.

[0122] Sequencer, generally refers to a device for determining the sequence of genetic material of a sample. Sequencers can function in a variety of ways and based on a variety of techniques, including sequencing by primer extension using labeled or unlabeled nucleotides, sequencing by synthesis, or pyrophosphate sequencing, for example, using any of the Sanger dideoxy method, nanopore or "NexGen" sequencing methods in the art (e.g., using MGI's sequencing platform, ROCHE 454 sequencing platform, ILLUMINA SOLEXA sequencing platform, LIFE TECHNOLOGIES / APPLIED BIOSYSTEMS' SOLID sequencing platform, PACIFIC BIOSCIENCES' SMRT sequencing platform, POLLONATOR Polony sequencing platform, COMPLETE GENOMICS sequencing platform, INTELLIGENT BIOSYSTEMS' sequencing platform, HELICOS sequencing platform, or any other sequencers and systems in the art).

[0123] Multiple staining imaging, which can be a multiple immunofluorescence staining (mIF) technique, can achieve in situ multi-target staining of tissues or cells through multiple staining cycles, thereby comprehensively studying cell composition, cell function, and cell-cell interactions. This method not only improves the sensitivity of detection, but also allows spatial distribution analysis of multiple biomarkers in the sample. It can also be a multiple fluorescent Western Blot, which uses different fluorescent labels to detect multiple target proteins in a protein sample. This method reduces background fluorescence and avoids signal leakage by optimizing the amount of sample and selecting appropriate fluorescent labels, thereby achieving simultaneous detection of multiple proteins. It can also be a multiple ion beam imaging (MIBI) technique and other methods that can quantify proteins at the single-cell or even subcellular structure level.

[0124] Iterative indirect immunofluorescence imaging (4i) is an imaging technique that can use a fluorescence microscope to display dozens of proteins in thin tissue sections at high resolution, developed by Professor Lucas Pelkmans of the University of Zurich.

[0125] Workstation is a high-performance computer system designed to meet the needs of professional users or specific industries. Workstations usually have powerful processing capabilities, high-quality graphics processing functions, and rich expandability, and can handle complex computing tasks, data analysis, graphics rendering, and engineering design, etc.

[0126] Sample detector is a device used to analyze and evaluate biological, chemical or physical samples. It can be at least one of an optical microscope, an electron microscope, a fluorescence microscope, a UV-Vis spectrophotometer, an infrared spectrometer, an NMR spectrometer, an HPLC, a GC, a MS, a flow cytometer, a gene sequencer, a mass spectrometer, a biochemical analyzer, a cell counter, a thermal cycler, an electrophoresis device, a biosafety cabinet, and a lyophilizer.

[0127] The above and other features and advantages of the present disclosure will become more apparent by describing in detail the non-limiting exemplary embodiments thereof with the accompanying drawings. The accompanying drawings, which constitute a part of this specification, illustrate embodiments of the present disclosure and together with the description, further serve to explain the principles of the present disclosure. For the purpose of clarity and a concise description, detailed descriptions of known functions and configurations will be omitted when it can obscure the subject matter of the present disclosure. It will be understood that the specific embodiments disclosed herein are illustrative in nature and are provided for explanatory purposes only and are not limiting.

[0128] The embodiments of the present disclosure provide assay systems for multiple detection modes involving optical scanning devices and related methods. The assay systems for multiple detection modes are particularly useful for biochemical sample detection, including a sample stage for holding a sample slide and an optical module for scanning a sample to be detected. The optical module has variable operation modes, which are related to the type of sample to be detected. Different operation modes of the optical module involve invoking different mode control parameters. It should be understood that in some specific applications, the optical module is configured to be relatively movable with the sample stage, and the operation of the liquid handling portion cooperating with the optical module, such as supplying fluid to the slide, the motion control module controlling the relative movement between the optical module and the sample stage, etc. can also have adaptive adjustments in different operation modes of the optical module, such as timing adjustment of the liquid supply and discharge speed of the liquid handling portion, changes in driving parameters of the motion control portion, etc.

[0129] In some embodiments, a set of parameters corresponding to different operation modes of the optical module can be predetermined and stored in the memory, for example, the optical module has any combination of a first operation mode (e.g., sequencing mode), a second operation mode (e.g., fluorescent staining mode), and a third operation mode (e.g., brightfield imaging mode). Correspondingly, first mode control parameters corresponding to the sequencing mode, second mode control parameters corresponding to the fluorescent staining mode, and third mode control parameters corresponding to the brightfield imaging mode are pre-stored in the memory for selective calling during actual operation. The first mode control parameters include laser power parameters, real-time focusing parameters, slide motion control parameters, fluid timing parameters, etc., wherein the real-time focusing parameters include, for example, adaptive algorithm supported real-time focusing module SNR parameters of the sequencing chip. The second mode control parameters include laser power parameters (e.g., 150 mW and 300 mW laser power) different from the first mode control parameters, focusing parameters (e.g., chip real-time focusing SNR parameters according to tissue autofluorescence), and fluid timing parameters corresponding to the second mode (e.g., corresponding to immunofluorescence staining fluid timing), and stitching parameters, etc. The third mode control parameters include, for example, laser power parameters of 100 mW and 200 mW laser, adaptive focusing parameters for image scoring recording according to cell / microorganism fluorescence intensity, and image sensor parameters corresponding to cell / microorganism fluorescence, etc. The selective calling of the above-mentioned control parameters is based on the type of sample to be detected in the sample slide, so that the system can give different real-time calling of mode control parameters according to the change of the sample to be detected, and timely switch the operation mode of the optical module, without additional equipment to realize multiple biochemical tests on biological samples.

[0130] As shown in FIGS. 1-2, in the assay system of various detection modes of one embodiment of the present disclosure, the sample slide 120 is used to carry the sample to be detected, and the sample slide 120 can be positioned on the sample stage 110 which is located below the objective lens of the optical module 110, for example, the sample slide can be fixed by vacuum adsorption or the like. The optical module 100 is movably arranged opposite to the sample stage 110. The optical module 100 has at least a sequencing mode and a fluorescent staining mode, and it should be understood that the optical module 100 can also have a third operation mode different from the sequencing mode and the fluorescent staining mode, for example, a brightfield imaging mode. The operation mode of the optical module 100 is related to the type of sample to be detected. The sample to be detected includes but is not limited to nucleic acid sequencing library, biological tissue sample, microbial sample or cell sample, etc.

[0131] In the sequencing mode, the optical module 100 scans the nucleic acid sequencing library through the objective lens and generates sequence information corresponding to the nucleic acid sequencing library; in the fluorescent staining mode, the optical module 100 scans the biological tissue sample through the objective lens and outputs an image corresponding to the biological tissue sample, microbial sample or cell sample. In the bright field imaging mode, the optical module 100 collects and outputs a bright field image of the sample to be detected. The optical module 100 comprises an image sensor and an image processing unit operatively connected to the image sensor, and the image processing unit receives a plurality of images corresponding to a plurality of FOVs generated by the image sensor in the fluorescent staining mode and integrates and splices the plurality of images.

[0132] As shown in FIG. 5A, the optical module 100 can comprise a first optical link 100a and a second optical link 100b independent of each other, and the sample stage 110 comprises at least two detection sites corresponding to the sample stage 110 for receiving a plurality of sample slides. The first optical link 100a comprises a first objective lens, and the second optical link 100b comprises a second objective lens. The first optical link is in the sequencing mode, and the second optical link is in the same or different mode as the first optical link. The operation modes of the two optical links can be independent of each other. It should be understood that the optical module 100 can further comprise a third optical link, and the first optical link, the second optical link and the third optical link are in any one of the sequencing mode, the fluorescent staining mode and a third operation mode at the same time.

[0133] Specifically, the light source module of the optical module comprises a plurality of light source elements such as an excitation light source L1 and a bright field light source L2. The plurality of light sources are selectively connected to, for example, a multiplexer MUX controlled by the controller 200, and selectively output light signals matching the operation mode of the optical module 100 to the sample to be detected. The sample to be detected generates corresponding detectable light signals under the irradiation of the light signals, for example, generates fluorescent signals under excitation of laser light in the sequencing mode or the fluorescent staining mode; generates bright field signals under reception of bright field light in the bright field imaging mode. The above-mentioned detectable light signals are transmitted through the objective lens and collected by the relevant optical sensor. In the sequencing mode, the optical sensor is associated with the corresponding base recognition device, and finally converts the detectable light signals into sequence information and outputs; and in the fluorescent staining mode or the bright field imaging mode, for example, the optical sensor is associated with the image processing device, and the detectable signals are output in the form of images. Specifically, the optical module comprises an image sensor and an image processing unit operatively connected to the image sensor, and the image processing unit receives a plurality of images corresponding to a plurality of FOVs generated by the image sensor in the fluorescent staining mode and integrates and splices the plurality of images.

[0134] The sample stage 110 is arranged to be relatively movable with respect to the objective lens to facilitate scanning of the objective lens over multiple detection sites of the sample to be detected. The relative movement between the sample stage 110 and the objective lens can be achieved by moving the sample stage itself, the objective lens, the entire optical stage, or any combination of the foregoing.

[0135] As shown in FIG. 3, the controller 200 includes a driving circuit 210 and a mode switching circuit 220, which are coupled to the optical module 100 to control the optical module 100 to collect data of the sample to be detected. The driving circuit 210 is selectively coupled to mode parameters corresponding to the operation mode of the optical module 100, which at least include first mode control parameters corresponding to the sequencing mode and second mode control parameters corresponding to the fluorescent staining mode. The mode switching circuit 220 is coupled to the driving circuit 210 to control the driving circuit based on the sample to be detected: when the sample slide is a sequencing chip, the mode switching circuit 220 associates the first set of mode parameters to the driving circuit 210, and when the sample slide is a tissue slide, the mode switching circuit 220 associates the second set of mode control parameters to the driving circuit 210. The mode switching circuit 220 selects the mode control parameters corresponding to the detected sample type after determining the type of the sample to be detected. Exemplarily, the mode control parameters can be stored in a memory or other similar storage device. The driving circuit 210 invokes the mode control parameters matched by the mode switching circuit 220 to load the corresponding circuits of the optical module 100. The host computer control software integrates the start-up parameters corresponding to different modes, and when the first set of mode parameters is started, the host computer can automatically switch / start the sequencing mode; when the second set of mode parameters is started, the host computer can automatically switch / start the fluorescent staining mode.

[0136] In some embodiments, the controller 200 further includes an identification circuit coupled to the mode switching circuit 220, which has an interface for identifying the type of the sample slide and / or the operation mode. The interface includes, for example, a user input port for receiving inputted slide type information and / or the operation mode. The sample slide has an identifiable type identifier, such as a barcode or a two-dimensional code; and the interface includes a scanning mechanism (e.g., a code scanning gun) for identifying the type of the sample slide according to the type identifier and transmitting the scanned information to the mode switching circuit 220.

[0137] As shown in FIG. 6, the disclosure discloses a detection method of the assay system of multiple detection modes described above. In step S11, the slide 120 is installed to the sample stage 110. In combination with FIG. 5, when multiple slides are involved in the detection, the assay system of multiple detection modes is provided with multiple sample stages and optical links, in which case, the first slide is installed to the first detection site of the sample stage; and the second slide is installed to the second detection site of the sample stage.

[0138] The detection method further comprises: loading a first sample to be detected to the first slide before or after the first slide is installed to the first detection site of the sample table, the first sample to be detected comprising a nucleic acid sequencing library, a biological tissue sample, a microorganism sample, or a cell sample. The detection method further comprises: loading a second sample to be detected to the second slide before or after the second slide is installed to the second detection site of the sample table, the second sample to be detected comprising a nucleic acid sequencing library, a biological tissue sample, a microorganism sample, or a cell sample. The types of samples to be detected on the first slide and the second slide can be different or the same. The optical module comprises a first optical link and a second optical link, and the detection method comprises: adjusting the relative position between the optical module and the sample table to align any one of the first slide and the second slide with any one of the first optical link and the second optical link.

[0139] Further, in step S12, the mode switching circuit determines the mode parameters selected to be coupled to the driving circuit, as described above, the mode parameters corresponding to the operation modes of the optical module, at least comprising a first mode control parameter corresponding to the sequencing mode and a second mode control parameter corresponding to the fluorescent staining mode. In further embodiments, the mode parameters can further comprise a third control mode parameter corresponding to a bright field imaging mode, for example.

[0140] In step S13, the driving circuit applies the mode parameters determined by the mode switching circuit to the optical module to switch the optical module between at least the sequencing mode and the fluorescent staining mode.

[0141] As shown in FIG. 7, the embodiments of the present disclosure also provide a mode switching method of the above-mentioned assay system of multiple detection modes. The mode switching method comprises: S21, identifying the type of sample to be detected, especially when the slide of the detection site on the sample table is replaced, for example, the assay system of multiple detection modes is provided with a sensor for monitoring the state of the slide of the detection site in real time or is monitored by scanning imaging and machine vision, when the slide of the detection site is replaced, the above-mentioned identification circuit is driven to perform the above-mentioned step S21.

[0142] Step S22 determines the mode parameters selected to be coupled to the driving circuit based on the type of sample to be detected, steps S231-232.

[0143] Step S24, when the sample to be detected is a nucleic acid sequencing library, the mode switching circuit associates the first group of mode parameters to the driving circuit, and the driving circuit drives the optical module into the sequencing mode; when the sample to be detected is a biological tissue sample, a microorganism sample or a cell sample, the mode switching circuit associates the second group of mode control parameters to the driving circuit, and the driving circuit drives the optical module into the fluorescent staining mode or a third operation mode different from the fluorescent staining mode and the sequencing mode. The third operation mode is the bright field imaging mode. When the types of samples to be detected are different, the real-time change of the type of sample can be detected by the above-mentioned identification circuit, and the new operation mode parameters are selected and called into the corresponding control loop of the optical module. In some applications, the calling of the operation mode parameters can occur in real time (but it can still depend on the system time delay or other delays), so that the operation mode of the optical system can be dynamically changed.

[0144] As shown in FIG. 8, in the assay system of various detection modes of one embodiment of the present disclosure, the sample slide includes a sample cavity for placing the sample to be detected, the sample cavity is a sealed cavity provided with an exchange hole for introducing fluid into the sample cavity or discharging fluid from the sample cavity, a liquid path module for introducing fluid into or discharging fluid from the sample cavity, including a reagent cavity and a pipeline connecting the reagent cavity and the sample cavity; and an imaging module for imaging the sample to be detected.

[0145] In the present disclosure, the sample cavity can be a detachable component to facilitate loading the sample to be detected therein, and it can be loaded before use. For example, the sample cavity is a detachable sample slide. The exchange hole includes an inlet hole and an outlet hole, which can be one or more, and optionally more than one. When fluid is introduced, the fluid enters the sample cavity through the inlet hole, and the air is discharged from the sample cavity through the outlet hole; when the fluid is discharged, the fluid is discharged from the sample cavity through the outlet hole, and the new fluid (liquid or gas) replaces the original fluid into the sample cavity. The entry and discharge of fluid can be performed by a pump, such as a micropump.

[0146] In the present disclosure, the reagent cavity can have multiple reagent cavities for containing multiple reagents, which can be the same or different, each of the multiple reagent cavities is connected to the sample cavity through a respective pipeline, and a gating valve is provided on the pipeline to introduce different reagents into the sample cavity as needed. The control module can be used to control the sequential introduction of multiple reagents from multiple reagent cavities into the sample cavity and the discharge of the reagents from the sample cavity.

[0147] In the present disclosure, the temperature of the fluid in the sample chamber and the reagent chamber can be controlled by the temperature control module, with the purpose of incubating the sample to be detected at a set temperature. Any method can be used to maintain the temperature of the fluid in the sample chamber and the reagent chamber, for example, by means of electrical heating and temperature-sensitive sensor to regulate the on and off of the electrical heating. It should be understood that the temperature control adjustment parameters of the temperature control module can be different when the sample to be detected is different.

[0148] In the fluorescence staining mode, the optical path schematic diagram of the optical module 100 is shown in FIG. 5B. The optical module 100 includes an objective lens 1001, an eyepiece 1006, fluorescence imaging cameras 1007-1008, and a light splitting / filtering element, etc. Specifically, the objective lens 1001 is arranged opposite to the sample slide 120 to collect the optical signal of the sample slide 120, and the fluorescence signal in the optical signal is transmitted by the light filtering assembly 1003. In the fluorescence staining mode, the light source module includes an epi-illumination light source L2a and a trans-illumination light source L2b, which can be selectively turned on by the user according to actual needs. The epi-illumination light source L2a is, for example, an epi-illumination LED light source, which is collimated and output, passes through the light filtering assembly 1003 into the objective lens 1001, uniformly illuminates the tissue section area of the sample slide 120 in the field of view of the objective lens 1001, and the fluorescent substance in the sample slide 120 is excited to emit fluorescence. The objective lens 1001 collects the fluorescence and collimates and outputs it, which is filtered by the light filtering assembly 1003 and then enters the tube lens, which is magnified and imaged on the sensor chip of the eyepiece 1006 or the fluorescence imaging camera 1007-1008. For example, a binocular beam splitter is also arranged between the eyepiece 1006 and the light filtering assembly 1003. During the camera exposure process, the sample slide 120 moves at a constant speed in a straight line, and the objective lens 1001 scans the imaging area of the biological tissue section. In order to ensure that the imaging area does not introduce excessive defocus when it changes, a focus detector can be additionally arranged to detect the defocus amount of the biological tissue section in real time, and a focusing structure is arranged to ensure that the tissue section is within the allowable defocus amount. Further, the epi-illumination light source L2a can selectively emit epi-fluorescence and epi-brightfield light, so the optical path shown in FIG. 5B can also be applicable to the brightfield imaging mode. In the epi-brightfield imaging mode, any of the cameras 1007-1008 can be replaced by a color camera, and the light filtering block 1003 is replaced by a brightfield light filtering block. Further, as shown in FIG. 5B, the brightfield imaging mode can also use trans-illumination: the trans-illumination LED light source L2b emits illumination light, which is collimated and output, enters the condenser lens after being reflected by the mirror, and the condenser lens focuses the light on the sample in the sample carrier 120 to obtain strong illumination. The brightfield light signal of the sample carrier 120 is magnified by the objective lens 1001, etc., and the image is on the sensor chip of the eyepiece 1006 or the camera 1007-1008 (in this case, any of the cameras 1007-1008 can be replaced by a color camera).

[0149] As shown in FIG. 5C, in the sequencing mode, the sample slide 120 includes a plurality of sample receiving sites 1201, and a DNA sample is placed in the sample sites 1201, which is capable of emitting a fluorescent signal. A light source (not shown) of the optical system emits a light signal toward the sample sites to stimulate the sample to generate a fluorescent signal. A light transmission layer (i.e., equivalent to a microlens) corresponding to each sample site is arranged to transmit the fluorescent signal of the sample site 1201 to the photoelectric sensor 1005 corresponding to each sample site. The photoelectric sensor 1005 collects the fluorescent signal and generates an analog electrical signal. Subsequently, the analog electrical signal is processed by the control circuit electrically connected to the photoelectric sensor 1005, and the type and / or sequence of the base contained in the sample are determined.

[0150] FIG. 8 shows a schematic diagram of a multi-detection mode assay system according to an exemplary embodiment of the present disclosure. The multi-detection mode assay system of FIG. 8 can include both sequencing mode and fluorescent staining mode, which further expands the function of the sequencer and realizes the multifunctionalization of the sequencer. As shown, the multi-detection mode assay system includes a slide for placing a sample to be detected, an optical module for optically scanning the sample to be detected, a liquid channel module for introducing and discharging liquid reagents to the sample to be imaged, a temperature control module for controlling the temperature of the sample chamber and the reagent chamber, and a control module for controlling the entire system. The sample chamber of the slide can be a sealed cavity provided with an exchange hole for introducing fluid into the sample chamber or discharging fluid from the sample chamber. Therefore, the exchange hole includes an inlet hole and an outlet hole. In some embodiments, the sample chamber is a detachable component, for example, a detachable sample slide can be used as the sample chamber. The optical module optionally has a light source module, an optical detector, a motion control and an automatic focusing mechanism, etc., for realizing the optical scanning function. By scanning the sample with a known focal length, at least one layer can be ensured to obtain a clear image through the shooting of several layers above and several layers below. The liquid channel module includes a reagent chamber and a pipeline connecting the reagent chamber and the sample chamber. The reagent chamber can have multiple chambers for containing multiple reagents, and each reagent chamber is connected to the sample chamber through a pipeline. Optionally, a gating valve is provided on the pipeline for selectively connecting the reagent chamber and the sample chamber. Sequentially supplying multiple reagents to the sample chamber can realize multiple incubations of multiple samples to be detected, and optionally cleaning between incubation with different reagents can be performed using water or buffer. Incubation and cleaning can be programmed, and the control module automatically performs fluid selection and fluid driving based on the set program to realize the sequential introduction of multiple reagents from multiple reagent chambers to the sample chamber and the discharge of the reagents from the sample chamber. The temperature control module can control the temperature of the sample chamber and the reagent chamber respectively, and the temperature of the sample chamber and the reagent chamber can be the same or different. In the case of multiple reagent chambers, the temperature of the multiple reagent chambers can be controlled respectively, and the temperature of different reagent chambers can be the same or different. The control module can realize automatic temperature control. The temperature control module can adopt a PID control mode to realize temperature adjustment, and the temperature change can be realized by a Peltier element built into the sample stage. The control module can be run by a software system and executed by a host computer, which is responsible for controlling the operation of the multi-detection mode assay system. The control module is used to control the liquid channel module to exchange reagents on the sample slide, and is used to control the microscope imaging module to image the sample slide, so that the incubation and imaging of the sample slide are performed in the required order. An editable configuration file allows the settings of the control module to be modified, and a customizable protocol file specifies the sequence of fluid processing, temperature setting and imaging steps.In addition, the position of the sample chamber can be moved (for example, by an x, y, z three-axis moving platform) by a moving platform to accurately position and move the sample on the chip, ensuring the superposition of multiple images and image stitching.

[0151] The scheme using the system of the present disclosure combines a microscope and automated operation of sample processing to be detected, which can visualize tens of thousands of features in millions of pixels of the sample. The present disclosure can implement a simple and effective, programmable signal amplification multi-channel, sensitive in-situ protein detection method. The present disclosure can make the imaging analysis from super-resolution research to centimeter-level tissue mapping work have higher throughput. These omics methods require accurate control of temperature, reagent application, and image acquisition parameters in the iterative chemical and imaging cycle of several days or weeks. Automatic execution of these methods enables robust and repeatable data generation.

[0152] In the assay system integrating multiple detection modes such as sequencing and fluorescent staining, the sample slide can be a sequencing slide for fixing nucleic acid detection library, or other slides for supporting, for example, biological tissue samples, and the sample slide is fixed on the sample stage. FIGS. 9A-9B show schematic diagrams of a sample slide according to an example embodiment of the present disclosure, FIG. 9A shows a sample slide assembly diagram, and FIG. 9B shows a sample slide assembly explosion diagram. FIGS. 10A-10D show schematic diagrams of preparing a tissue section adhered to a sample slide of an example embodiment, fixing the tissue section to facilitate subsequent incubation, and preventing the tissue section from moving in the liquid during incubation and washing of the tissue section. Among them, FIG. 10A shows the process of adhering the tissue section to the slide; FIG. 10B shows the state after adhering the tissue section to the slide; FIG. 10C shows adhering the double-sided tape to the slide; and FIG. 10D shows completing the assembly of the sample slide by covering the frame with the cover glass. The sample slide is fixed on the chip operation stage. The present disclosure is suitable for a double-layer slide flow channel design of a tissue section in the form of a paraffin tissue section, a frozen tissue section, etc. In an embodiment, after pretreatment, the tissue section is transferred to the slide and fixed with the slide, then the flow channel cut from the double-sided tape is pasted on the slide, and finally the cover glass is covered on top (alternatively, the double-sided tape can be pasted on the cover glass first, and then pasted on the slide). Through the above method, a three-layer structure flow channel chip is formed. The sample slide can be fixed on the fixture base. FIG. 11 shows a schematic diagram of a sample slide fitting and fixing jig. The chip is placed in the chip fixing position of the base, and pressure is applied downward to the compression block, thereby compressing the chip. In this embodiment, the base is made of engineering plastic and has a certain hardness; the compression block is made of silicone and forms a compression effect on the chip with the base, but does not damage the chip at the same time.

[0153] As described above, the multi-detection mode assay system of the present disclosure integrates sequencing mode and multi-mode detection schemes such as fluorescent staining mode to form a staining imaging sequencing multifunctional platform. The multi-detection mode assay system can expand the application of the sequencer, realize the integration of different applications, and improve the use value of the instrument. By introducing the multi-detection mode assay system of the present disclosure, the present disclosure converts the sequencer into a multifunctional platform compatible with protein tissue staining and imaging in addition to gene sequencing functions. Through the fluorescent staining operation designed for the instrument, the experimental design includes synchronous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity for extended experiments. Similar to existing sequencers, this scheme can realize the function of iterative indirect immunofluorescence imaging (4i) in a complex multi-day continuous workflow cycle without manual intervention. At present, the system of the present disclosure can perform a highly automated 4i method during the fluorescent staining imaging process, and is compatible with most off-the-shelf antibodies on the market. After multiple cycles of staining, imaging, and antibody elution, a highly multiplexed map of animal or plant cell types and pathological features is finally constructed. In addition to indirect immunofluorescence imaging, the scheme of the present disclosure is also applicable to Vectra multispectral imaging, Lunaphore microfluidic liquid exchange system, CellDive platform, CyCif platform, ZellScannerONE instrument platform, Immuno-Saber platform, PhenoCycler platform, and InSituPlex technology. In the present disclosure, the optical system is modified to realize a multiplexing scheme of more than 6 markers per round through multispectral imaging. With the development of imaging and spectroscopy, it is possible to demix the image, and different biological molecules are labeled with multiple dyes. Even if there is very obvious spectral overlap, each optical signal can be separated from each other through spectral demixing.

[0154] The scheme of the present disclosure can be applied to immunofluorescence imaging, nucleotide-coupled antibody fluorescence imaging, RNA in situ hybridization fluorescence imaging (corresponding to RNAScope hybridization signal), and supports in situ sequencing of nucleotide extension on tissue samples. The multiplex biochemical detection of the present disclosure is suitable for various antibody fluorescent staining reagent schemes, including 4i based on fluorescently labeled secondary antibody amplification, TSA signal amplification, fluorescently labeled nucleotide-coupled antibody CODEX (co-detection by indEXing), etc. Among them:

[0155] Fluorescent labeling amplification technology: circulating indirect immunofluorescence imaging (4i for short) is the first imaging technology that can provide multiplexing from the perspective of tissue to organelle. In the same experiment, it can obtain correlated multiplexing information at the tissue, cell, and subcellular levels. Immunofluorescence (IF) is used to visualize and locate proteins in biological samples using antibodies. Standard IF can usually mark 2-3 proteins. 4i uses existing antibodies and conventional fluorescence microscopes to visualize IF 10 times the number of proteins through repeated removal of antibodies and hybridization.

[0156] TSA (Tyramide Signal Amplification) tissue section multiplex labeling scheme of tyramide signal amplification technology, which uses horseradish peroxidase (HRP) to mark target proteins or nucleic acids in situ at high density, improves detection sensitivity, and greatly improves signal-to-noise ratio even up to thousands of times. It can use the same animal-derived primary antibody to perform fluorescent multiplex labeling of multiple markers on the same tissue section. At the same time, due to its excellent signal amplification performance, it can increase the signal intensity by 10-100 times, greatly improving the detection sensitivity of weak signals and proteins that are not easy to mark.

[0157] The CODEX platform combines oligonucleotide labeling (Barcode) and microfluidic automatic staining technology, which can realize the detection and deep analysis of more than 50 protein markers in the same sample, and can provide high-resolution images at the single-cell level. The core design principle of CODEX is to label specific oligonucleotide "barcode labels" (Barcode) on each antibody, rather than directly labeling fluorescent dyes. The fluorescent dyes required for imaging are specifically bound through oligonucleotide sequences complementary to Barcode, so that CODEX breaks through the limitation of the number of visible light spectrum fluorescence imaging channels, and easily realizes the simultaneous detection and analysis of 50 or more protein indicators.

[0158] The scheme of the present disclosure is also applicable to RNA and DNA targets, to supplement single-cell RNA sequencing analysis with functional information of protein expression, and will be proven to have wide potential application value, including tissue mapping, tumor and disease mapping. In situ single-cell verification for bulk detection, such as flow cytometry or mass cytometry, and CITE-Seq (single-cell sequencing of cell surface proteins and RNA at the same time), as well as digital pathology and biomarker screening and discovery.

[0159] The scheme of the present disclosure is also applicable to the use of DNA or RNA probes to detect the location of another complementary strand in bacteria or other eukaryotic cells.

[0160] RNA in situ nucleic acid hybridization is also known as RNA in situ hybridization histochemistry or RNA in situ hybridization. This technique refers to a kind of in situ hybridization technology for detecting RNA expression in cells and tissues by using cRNA or oligonucleotide probes. Its basic principle is: under the condition that the structure of cells or tissues is kept unchanged, a labeled known RNA nucleotide fragment is combined (hybridized) with the corresponding gene fragment in the cells or tissues to be tested according to the base pairing principle in nucleic acid hybridization, and the formed hybrid is observed under an optical microscope or an electron microscope after color development to detect the corresponding mRNA, rRNA and tRNA molecules in the cells. The RNA in situ hybridization technology has been continuously improved, and its application field has far exceeded that of the DNA in situ hybridization technology. Especially in gene analysis and diagnosis, it can be used for qualitative, positional and quantitative analysis, and has become the most effective molecular pathology technology. At the same time, it has shown an important direction of molecular biology in analyzing low-abundance and rare mRNA expression. The detection of nucleic acid targets can use the method of probe hybridization, and the oligonucleotide probe can reversibly hybridize to the target nucleic acid and undergo extension and ligation reaction. In the detection reaction, when the probes bind to the target in the correct direction, they can undergo extension and ligation reaction, thereby generating a ligated oligonucleotide product. The presence or amount of the ligated oligonucleotide product can then be measured to determine the presence of the target of interest. Among them, the oligonucleotide probe can be directly linked to a detectable label, such as a fluorescent label, an electrochemical label, a magnetic bead, a nanoparticle, biotin, etc. for quantifying or detecting the ligated oligonucleotide product. Alternatively, the oligonucleotide probe can not be linked to a detectable signal, and the oligonucleotide probe has specificity for the target of interest and generates a ligation product at each target site, and an amplification reaction for signal amplification can be performed, generating multiple copies of the ligation product, and a signal probe is used to hybridize to the amplification product, thereby amplifying the detectable signal. In some cases, the nucleic acid target is an RNA target, and the RNA target can be reverse transcribed to generate a cDNA. The cDNA can then be amplified or can be contacted with a probe (e.g., a padlock probe). The probe can hybridize to the cDNA. In some cases, the nucleic acid target is a DNA target, and the DNA target can be subjected to amplification or can be contacted with a probe (e.g., a padlock probe). For example, the DNA target can be directly amplified by amplification primers. For another example, the padlock probe can be contacted with the DNA target and hybridized to the DNA target. The padlock probe can then be circularized and amplified. The amplification product or amplicon can directly bind to a signal probe for in situ signal detection. FIG. 15 shows in situ mRNA detection data obtained using RNA in situ hybridization sequencing technology.

[0161] In some embodiments, the signal probe can be a sequence-specific oligonucleotide probe that has optical activity when hybridized to a nucleic acid target or derivative thereof (e.g., amplification product). The probe can be linked to any of the optical active (e.g., dyes) described herein, and can also include a quencher capable of blocking the optical activity of the associated dye. Non-limiting examples of probes that can be used as reporters include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

[0162] Figure 12 shows a schematic diagram of a fluidic module of a multiplex detection assay system according to one embodiment of the present disclosure. As shown, the multiplex detection assay system includes a plurality of reagent cartridges 1 for storing a plurality of reagents. The reagent cartridges 1 are in fluid communication with a sample slide 2 as a sample chamber through lines with a gate valve 3 for gating the reagents in different reagent cartridges 1. The sample slide 2 is also in fluid communication with a waste tank 5 through lines for draining the reagents from the sample slide 2. A syringe pump 4 is installed on the recovery line connecting the waste tank 5, which provides power for the reagents to be drawn from the reagent cartridges 1, to enter the chip 2 through a precision fluidic system, to act on the tissue in the chip 2, and then to be drawn out of the chip 2 and finally into the waste tank 5. The microfluidic system can ensure fast reaction and high stability and repeatability of the target and the reagent. In one example of the present disclosure, for multiplex biochemical detection in the fluorescent staining mode, sample preparation can include the following steps: a) set the slide so that the sample is firmly bonded to the slide; b) after the sample is mounted, it can be pre-processed by traditional methods before being loaded into the machine; c) after being loaded into the machine, heating can be performed as needed through a temperature control system, and antigen retrieval can be performed under strong acid or strong base conditions. The purpose of antigen retrieval is to fully expose the antigens cross-linked with paraffin so that the antibody can bind to the relevant antigens. The acid, base, heat, or pressure used should be adjusted according to the specific antibody; d) use 0.5% Triton X-100 to incubate the sample for 15 minutes to punch holes in the cell membrane. The purpose of punching holes in the cell membrane is to allow the antibody to enter the cell and bind to the relevant antigens. In the present disclosure, the entire sample preparation process can be reduced from 300 minutes to about 180 minutes. After loading the sample, the following multiple fluorescent staining pretreatment can be performed: a) use PBS for flow cleaning (time: 100 seconds, flow rate: 1200 microliters per minute); b) use blocking reagent (such as 10% BSA, 5% Goat Serum, or 10% skim milk, etc.) for 30 minutes of blocking treatment; c) use PBS for flow cleaning (time: 100 seconds, flow rate: 1200 microliters per minute); d) add imaging buffer (Trolox, N-acetyl cysteine, DTT, etc. antioxidant reagent solution); e) perform full slide scanning to obtain the autofluorescence background for correction. The method of the present disclosure can reduce the time to 5.3 hours or even less, while the corresponding operation of traditional fluorescent staining takes a full process of 8 hours or even longer. Moreover, most of the operations of the method of the present disclosure are completely automated by the machine without the need for human intervention.

[0163] The following exemplary gives three different scheme examples of multiplex fluorescent staining.

[0164] Example One

[0165] 1) Select the fluorescent staining mode in the multiplex detection instrument, and perform fluorescent staining detection.

[0166] Immunofluorescence detection is a secondary antibody amplification method:

[0167] i. Flow washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0168] ii. Add 1% BSA solution diluted primary antibody (1:10-1:400), incubate at 37 degrees Celsius for 15-45 minutes;

[0169] iii. Flow washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0170] iv. Add 1% BSA solution diluted fluorescently labeled secondary antibody (1:10-1:400), incubate at 37 degrees Celsius for 10-30 minutes;

[0171] v. Flow washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0172] vi. Add imaging buffer (Trolox, N-acetyl cysteine, DTT, etc. Antioxidant reagent solution);

[0173] vii. Whole slide scanning of the specific wavelength of the fluorescently labeled secondary antibody is performed;

[0174] viii. Antibody elution using antibody elution solution (weakly acidic 2ME+SDS+TrisHCl solution, acidic glycine+SDS solution or acidic glycine+urea+GC+TCEP solution, etc.) in temperature control (25-56 degrees Celsius) is performed;

[0175] ix. Flow washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0176] x. Again, use blocking reagents (such as 10% BSA, 5% Goat Serum or 10% skim milk, etc.) for 30 minutes of blocking treatment;

[0177] xi. Repeat steps i. to x. for multiple rounds of staining.

[0178] Example Two

[0179] 2) Select the fluorescence staining mode in the multi-mode detection instrument, and perform fluorescence staining detection.

[0180] Immunofluorescence staining is a fluorescently labeled primary antibody method:

[0181] i. Flow washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0182] ii. Add fluorescently labeled primary antibody (1:10-1:400) diluted in 1% BSA solution and incubate for 15-45 minutes at 37 degrees Celsius;

[0183] iii. Flow through wash with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0184] iv. Add imaging buffer (solution of Trolox, N-acetyl cysteine, DTT, and other anti-oxidant reagents);

[0185] v. Perform full-scan imaging of fluorescently labeled primary antibody at specific wavelength;

[0186] vi. Perform antibody elution using antibody elution solution (weakly acidic 2ME + SDS + Tris HC1 solution, acidic glycine + SDS solution, or acidic glycine + urea + GC + TCEP solution, etc.) for 30 minutes in temperature control (25-56 degrees Celsius). Alternatively, perform fluorescence quenching using quenching reagent (0.1 M sodium bicarbonate + 3% hydrogen peroxide, sodium borohydride, 24 mM sodium hydroxide + 4.5% hydrogen peroxide, etc.);

[0187] vii. Flow through wash with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0188] viii. Perform blocking treatment again using blocking reagent (e.g., 10% BSA, 5% Goat Serum, or 10% skim milk, etc.) for 30 minutes;

[0189] ix. Repeat i. through viii. for multiple rounds of staining and imaging.

[0190] Example Three

[0191] 3) Select fluorescent staining mode in multi-mode detection instrument and perform fluorescent staining detection.

[0192] Immunofluorescent staining is nucleotide conjugated fluorescence:

[0193] i. Flow through wash with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0194] ii. Add nucleotide conjugated primary antibody (1:10-1:400) diluted in 1% BSA solution and incubate for 15-45 minutes at 37 degrees Celsius;

[0195] iii. Add PFA or other amine fixing solution for antibody-sample fixation;

[0196] iv. Flow through wash with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0197] v. The nucleotide coupled on the primary antibody can be amplified by RCA, PER (primer replacement method) and other amplification methods;

[0198] vi. Flowing washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0199] vii. The corresponding fluorescently labeled nucleotide double strand is added to the solution (PBS, TBS, etc.), the sample is added, and incubated for 30 minutes, and stepwise cooling incubation can be performed to enhance specificity;

[0200] viii. Flowing washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute);

[0201] ix. Add imaging buffer (Trolox, N-acetyl cysteine, DTT, etc. Antioxidant reagent solution);

[0202] x. Full-slice scanning of fluorescent specific wavelength is performed;

[0203] xi. Double-stranded DNA can be eluted by high-temperature unwinding, double-stranded DNA degradation enzyme degradation, DNA unwinding enzyme unwinding elution, etc. to elute the fluorescently labeled nucleotide double strand;

[0204] xii. Repeat vi. to xi for multiple rounds of staining and photographing.

[0205] In the multiple fluorescence staining scheme, the present disclosure can add a reducing agent to the reagent, so that the antibody is more easily eluted from the cross-linking caused by the photographing.

[0206] In the multi-mode detection instrument, the sequencing mode is selected, and the nucleic acid sequence detection is performed.

[0207] After the multi-mode detection mode, the instrument can be switched to the sequencing mode to sequence the prepared nucleic acid library. The specific steps are as follows:

[0208] i. Deep cleaning of the multi-mode detection instrument;

[0209] Sequencing is performed using DNBSEQ-G400 PE150 sequencing kit, and Table 1 is the off-machine report of sequencing the nucleic acid library in the four lanes of the chip after the staining and photographing mode.

[0210] Table 1

[0211] The results of the staining and photographing are shown in FIG. 13, which are the results of multiple fluorescence staining of mouse brain section tissues photographed by using the multi-mode detection instrument imaging platform. Among them, the green area: GFAP antibody staining, the red area: fibronectin antibody staining. The imaging parameters are set as follows: laser intensity: red—300 mW, green—150 mW; exposure time: 40 ms.

[0212] Example Four

[0213] The multi-mode detection instrument can perform mRNA in situ hybridization detection. The following exemplary scheme based on mRNA in situ hybridization is given.

[0214] In the multi-mode detection instrument, the fluorescence staining mode is selected for fluorescence staining detection.

[0215] The in situ detection method is mRNA in situ hybridization detection.

[0216] 1) Primary probe binding:

[0217] i. Perform flow washing with 2XSSC buffer (time: 100 seconds, flow rate: 1200 microliters per minute);

[0218] ii. Add Primary probe (9 nM) diluted with 10-40% formamide and 0-10% dextran sulfate solution, and incubate at 37 degrees Celsius for 3 hours;

[0219] iii. Perform flow washing with 2XSSC buffer (time: 100 seconds, flow rate: 1200 microliters per minute);

[0220] iv. Add Secondary probe (20 nM) diluted with 10-40% formamide and 0-10% dextran sulfate solution, and incubate at 37 degrees Celsius for 1 hour;

[0221] v. Perform flow washing with 2XSSC buffer (time: 100 seconds, flow rate: 1200 microliters per minute);

[0222] vi. Add Tertiary probe (20 nM) diluted with 10-40% formamide and 0-10% dextran sulfate solution, and incubate at 37 degrees Celsius for 1 hour;

[0223] vii. Repeat steps iii-vi for signal amplification;

[0224] viii. Perform flow washing with 2XSSC buffer (time: 100 seconds, flow rate: 1200 microliters per minute);

[0225] ix. Add the Readout probe (10 nM) diluted in 10-40% formamide, 0-10% dextran sulfate solution, and incubate at 37 degrees Celsius for 15 minutes;

[0226] x. Perform flow washing with 2X SSC buffer (time: 100 seconds, flow rate: 1200 microliters per minute);

[0227] xi. Add imaging buffer for imaging.

[0228] The results are shown in FIG. 15, which shows the results of multiplex fluorescence staining of mouse tumor model section tissues purchased from Wuhan Saiver using the imaging platform of the multi-mode detector. Among them, the green area: Cd8 mRNA, the red area: Ki67 mRNA. Imaging settings: laser intensity: red - 300 mW, green - 150 mW; exposure time: 40 ms.

[0229] The system and method of the present disclosure liberate manpower by realizing a high-automation staining + photographing full process that can be unattended; compared with the staining and photographing separation instrument of the prior art, the staining and photographing functions can be integrated in the instrument; the scheme of the present disclosure can accurately control the amount of reagent, so that stable data of multiple rounds of quantification can be generated; the scheme of the present disclosure supports single section multiplex immunofluorescence staining, and is compatible with various biochemical staining schemes; the scheme of the present disclosure can greatly optimize the reagent reaction time.

[0230] In one example of the present disclosure, for analysis and detection in the cell or microbial mode, the chip preparation can include the following steps: a) set the sequencing chip, and fix the capture probe on the chip surface; b) load the cell suspension to the chip surface, perform fixation treatment and scanning and photographing (fluorescence photographing or bright field photographing can be selected as needed); c) perform permeabilization treatment on the cells, so that the mRNA in the cells can pass through the cell membrane and be captured by the capture probe on the chip surface; d) perform reverse transcription and sequencing library construction on the chip surface; e) switch the multi-mode detector to the sequencing mode, and obtain the transcriptome data by sequencing; f) integrate and analyze the transcriptome data and the cell photographing data to obtain the single cell transcriptome data. The method of the present disclosure is automatically operated by the multi-mode detector, and does not require personnel participation.

[0231] In one example of the present disclosure, for analysis and detection of cells or microorganisms, the chip preparation can include the following steps: a) set up a sequencing chip, and fix capture probes on the chip surface; b) extract cell nuclei from cells or tissues; c) add transposition complex and incubate with the cell nuclei to perform a transposition reaction; d) load the cell nucleus suspension onto the chip surface, perform fixation treatment, and scan and take pictures (fluorescence or bright field photography can be selected as needed); e) lyse the cell nuclei, and capture the genomic fragments broken by the transposition complex by the capture probes on the chip surface; f) perform amplification extension and sequencing library construction on the chip surface; g) switch the multi-mode detection instrument to sequencing mode, and obtain epigenome data by sequencing; h) integrate and analyze the epigenome data and the cell nucleus picture data to obtain single-cell epigenome data. According to the experimental purpose, single-cell transcriptome data and epigenetic data can be integrated and analyzed to obtain single-cell multi-omics analysis data.

[0232] The following exemplary gives a scheme example of cell or microorganism analysis and detection, as shown in FIG. 16, and the analysis process of the cell or microorganism analysis and detection result and sequencing result includes: S31, scanning the cell chip and taking pictures to obtain scanning images; S32, obtaining sample nucleic acid sequence information in sequencing mode; S33, analyzing and integrating the sequencing data and the cell images; S34, performing cluster analysis on the cell transcriptome data to obtain single-cell transcriptome data.

[0233] Embodiment five

[0234] Start the multi-mode detection analyzer, and select the cell or microorganism analysis mode.

[0235] 1. Prepare the sample phase suspension: 10000 cell nuclei are taken to prepare the cell phase, and the preparation system is (100 μL system).

[0236] 2. Sample loading: fix the sequencing chip on the stage, and load the cell suspension onto the chip.

[0237] 3. Fixation: load pre-cooled methanol for low-temperature incubation for 20 minutes, fix the cells on the chip surface, and scan and take pictures.

[0238] 4. Permeabilization: dilute 0.01M HCl by 10 times to obtain a working solution concentration (the working solution volume is 100 μL / chip), load onto the chip surface, and incubate at 37°C for 0-30 minutes.

[0239] 5. Reverse transcription: perform reverse transcription reaction (Huada Stereo-seq transcriptome kit, item number: 101KT114) after hybridization and capture of the mRNA in the cells by the capture probes fixed on the chip surface.

[0240] 6. Library construction: perform circularization and rolling circle amplification reaction on the chip surface, or sequencing library.

[0241] 7. Switching the multi-modal detection analyzer to sequencing mode, performing sequencing reaction, and obtaining transcriptome data.

[0242] 8. Integrating the transcriptome data and the cell images collected in step 3) for analysis, obtaining single cell transcriptome data (FIG. 17), wherein FIG. 17 is a partial chip image showing a large number of single cells distributed on the chip. One of the cells is circled, and the average gene number and UMI number captured by the cell can be seen.

[0243] FIG. 14 is a computer system for implementing the assay system or method in one embodiment of the present disclosure. The control system is implemented by a computer system programmed or otherwise configured to control the optical system, fluidic system, detection system, and analysis system to process the sample to be tested according to the established procedures using the methods of the present disclosure. The computer system can regulate various aspects of the processing of the various systems of the present disclosure, for example, processing instructions in a processor, contacting reagents or buffers with the sample to be tested, performing reactions on the surface of the slide, and controlling the optical system and detection system to collect signals. The computer system can be an electronic device integrated with the multi-modal assay system or a computer system remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0244] The computer system includes a central processing unit (CPU, also "processor" and "computer processor" herein) which can be a single core or multiple core processor, or a plurality of processors for parallel processing. The computer system also includes memory or memory location 310 (e.g., random access memory, read only memory, flash memory), electronic storage unit (e.g., hard disk), communication interface (e.g., network adapter) to communicate with one or more other systems, and peripheral devices, such as cache, other memory, data storage, and / or electronic display adapters. The memory, storage unit, interface, and peripheral devices are in communication with the CPU through a communication bus, such as a motherboard. The storage unit can be a data storage unit (or data repository) for storing data. The computer system can be operatively coupled to a computer network ("network") by means of the communication interface. The network can be the Internet, an internet and / or an extranet, or an intranet and / or extranet that in turn can be in communication with the Internet. The network in some cases is a telecommunication and / or data network. The network can include one or more computer servers, which can implement a distributed computing solution, such as a cloud-computing solution.

[0245] A CPU can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a memory location, such as a storage in the memory. The instructions can be directed to the CPU, which can subsequently program or otherwise configure the CPU to implement a method of the present disclosure. Examples of actions performed by the CPU can include fetch, decode, execute, and write back. The CPU can be part of a circuit, such as an integrated circuit.

[0246] The one or more processors 102 and / or other data processing circuitry described above can be referred to herein generally as "data processing circuitry." The data processing circuitry can be embodied in whole or in part as software, hardware, firmware, or any combination thereof. Furthermore, the data processing circuitry can be a single standalone processing module, or incorporated in whole or in part within any of the other elements of the computer terminal 10 (or mobile device). As referred to in embodiments of the present disclosure, the data processing circuitry functions as a processor to control, for example, selection of the variable resistance terminal path connected with the interface.

[0247] The memory 104 can be used to store software programs and modules of application software, such as program instructions / data storage means corresponding to the method of detecting a sample in embodiments of the present disclosure. The processor 102 can execute various functional applications and data processing by running the software programs and modules stored in the memory 104, i.e., implement the vulnerability detection method of the application program described above. The memory 104 can include a high-speed random access memory, and can further include a non-volatile memory, such as one or more magnetic storage devices, flash memories, or other non-volatile solid-state memories. In some examples, the memory 104 can further include a memory disposed remotely with respect to the processor 102, which can be connected to the computer terminal 10 through a network. Examples of the network include, but are not limited to, the Internet, an intranet, a local area network, a mobile communication network, and combinations thereof.

[0248] The transmission device 106 is configured to receive or send data via a network. Examples of the network include, but are not limited to, a wireless network provided by a communication provider of the computer terminal 10. In one example, the transmission device 106 includes a network adapter (NIC), which can be connected to other network devices through a base station to communicate with the Internet. In one example, the transmission device 106 can be a radio frequency (RF) module configured to communicate with the Internet in a wireless manner.

[0249] The display can be, for example, a liquid crystal display (LCD) in a touch screen type, which can enable a user to interact with the user interface of the computer terminal 10 (or mobile device), but is not limited thereto.

[0250] In some embodiments, the control system comprises at least one interaction unit for obtaining a first instruction of a user selecting at least one of the at least two omics analyses.

[0251] In some embodiments, the omics analysis selected by the user is based on the detection results of different detection modes that have been obtained, and different omics analysis contents are selected for different detection modes, for example, sequencing data can be used for genomic analysis or transcriptomic analysis, tissue sample staining data can be used for pathological analysis, proteomic analysis or spatial analysis, etc.

[0252] In some embodiments, the interaction unit can be a physical computer, a keyboard, a mouse, a touch display screen, etc., or a virtual virtual machine for the purpose of machine and user information interaction, without limitation to specific forms. In some embodiments, the unit for the multi-mode detector to interact with the user is a first interaction unit, and the unit for the workstation to interact with the user is a second interaction unit. The multi-mode detector and the workstation can communicate through a local area network transmission mode, for example, through a network hard disk directly connected by a network cable using a TCP / IP protocol, but are not limited thereto.

[0253] Among them, omics analysis refers to analysis of at least one of genome, cell group, proteome, metabolome, transcriptome, and epigenetic group. Taking genomic and proteomic analysis as an example, the inventive concept of one embodiment of the present disclosure is introduced.

[0254] Specifically, the first instruction can be an instruction of the user clicking a control of different software or function, for example, including base recognition, image conversion, and analysis format file such as Cal file, BCL file or FastQ file; or an instruction of clicking a control of bright field or dark field staining software or function.

[0255] The stage can be a platform for realizing temperature control, chip or chip and chip frame placement, chip fluid injection or suction, and data collection in cooperation with the optical unit.

[0256] Specifically, the fluid system further comprises a reaction unit for performing biochemical reactions on the sample.

[0257] The reaction unit can realize biochemical reactions for sequencing, and can also realize biochemical processes for immunohistochemistry, multiplex staining imaging.

[0258] Specifically, the device further comprises at least one processor coupled to the interaction unit, the stage and the reaction unit, for:

[0259] According to the first instruction, the user is instructed to input initial information, the user input mode includes at least one of keyboard input, touch screen input, gesture input, handwriting input, mouse input, code scanning input, camera input, radio frequency identification input, voice input and brain-computer interface input, and the initial information includes at least one of chip type, sequencing scheme, staining scheme, kit number and confirmation information.

[0260] Wherein, the code scanning input can be an external code scanning gun inputting the identification code on the slide or chip, or an optical unit inputting the identification code on the slide. Here, no limitation is made.

[0261] Wherein, the sequencing scheme can be any one of Sanger sequencing, NGS sequencing, single molecule sequencing and nanopore sequencing, but is not limited thereto.

[0262] Wherein, the staining scheme refers to a staining scheme including antibody name, wavelength and staining time, or a nucleic acid probe fluorescence detection scheme.

[0263] Wherein, the kit number refers to different codes of different types of kits.

[0264] Wherein, the confirmation information refers to confirming the information input by the user, such as confirming the input antibody, time and other information during protein analysis, so as to avoid the user from finding the input error and clicking to directly enter the next step, thereby effectively preventing mistakes.

[0265] The processor is further configured to control the reaction unit to perform biochemical reactions according to the initial information.

[0266] The processor is further configured to control the interaction unit to feed back the information being processed by the user.

[0267] In some embodiments, during the sequencing process or the staining process, the user can obtain images in stages, for example, a sequencing image or result is displayed once after a preset number of cycles are sequenced, or only a "sequencing in progress" interface is prompted without displaying the sequencing image or result. For another example, the image formed after each round of bright field or dark field staining is displayed through the interaction unit. For another example, the countdown time of completing the biochemical reaction is displayed. In this way, the interaction unit can feed back the information being processed by the user.

[0268] In another embodiment, for the technical route of bright field or dark field staining, an image after this round of staining can appear after each round of scanning. If the user judges that the result or quality of the staining does not meet the expectation, the biochemical reaction process can be interrupted or terminated.

[0269] The processor is further configured to generate a first processing result based on the reaction unit completing the biochemical reaction.

[0270] Specifically, the first processing result can be a Cal file, a BCL file, a FastQ file, a report, a quality control report, etc. after sequencing is completed, but is not limited thereto. It can also be a highly multiplexed map of cell types and pathological features of animals or plants after multiple cycles of staining, imaging, and antibody elution. It can also be a result produced after processing a transcriptome, a metabolome, and an epigenetic group, such as expression statistics, differential expression analysis, functional enrichment analysis, metabolite identification, metabolic pathway analysis, methylation level analysis, ChIP-seq analysis, functional and pathway analysis data, information or reports.

[0271] The processor is further configured to control the interaction unit to feed back the first processing result to the user.

[0272] Specifically, the first processing result is data, information or a report, which can be fed back to the user through a display screen, voice prompts, and brain-computer interface output, but is not limited thereto.

[0273] In some embodiments, integrated circuits such as CMOS FET and biosensors are often integrated on expansion cards, such as Peripheral Component Interconnect (PCI) cards. These expansion cards can be easily installed in computing devices, providing users with additional processing power and sensor data acquisition functions. Integrated circuits can also include field programmable gate arrays (FPGA) and application specific integrated circuit (ASIC) chips, which are key components for high-performance computing. FPGA is known for its flexibility and reconfigurability, and can be adjusted in real time according to changing application requirements. ASIC chips are optimized for specific tasks to improve execution efficiency and reduce energy consumption.

[0274] In other embodiments, with the development of cloud computing technology, local computing resources can be combined with cloud platforms to form a hybrid computing environment. Cloud platforms provide almost unlimited storage space and computing resources, which can be dynamically expanded according to demand. Local computing resources are responsible for processing tasks with high real-time requirements, while cloud platforms store large amounts of data and process large-scale computing tasks that can be processed in parallel. Through this hybrid computing mode, the system can more effectively manage and allocate computing loads to ensure optimal use of resources. For example, cloud platforms can provide necessary support for large-scale data analysis and storage-intensive applications. For tasks that require fast response and processing, local FPGA / ASIC chipsets and CPUs / GPUs can provide the required computing power.

[0275] In some embodiments, the at least one interaction unit further comprises a second instruction for obtaining the user input.

[0276] In some embodiments, the first instruction and the second instruction input by the user can be acquired through one interaction unit, or the first instruction and the second instruction input by the user can be acquired through different interaction units respectively, which can be set according to actual needs, and depends on hardware design, which will not be described here.

[0277] In some embodiments, the second instruction can be an instruction of clicking primary analysis, secondary analysis, tertiary analysis, or can be a control of clicking a software or function of cell analysis or protein analysis, but is not limited thereto.

[0278] Specifically, the processor is further configured to instruct the user to input analysis information according to the second instruction, and the analysis information includes at least one of opening a to-be-analyzed file, selecting an interested region, and selecting an analysis target.

[0279] The to-be-analyzed file can be a sequencing report or a sequencing file after sequencing, or can be a protein or cell image set after multiple rounds of staining. Opening the to-be-analyzed file can include a process of loading colors to the protein or cell image set. Selecting the interested region can determine the range of cell segmentation and analysis, which can be automatically selected or manually selected according to user needs. Selecting the analysis target can be selecting different markers, determining the number and probability of cells corresponding to the markers in the finally generated table, or can be a control displayed on an interface corresponding to primary analysis, secondary analysis, tertiary analysis, diagnostic analysis of the sequencing data, conversion of the Cal file or the BCL file to the FastQ file, so as to determine what kind of analysis is performed subsequently.

[0280] Specifically, the processor is further configured to analyze the first processing result according to a third preset method to generate a second processing result, and the third preset method includes at least one of converting the Cal file or the BCL file to the FastQ file, primary analysis, secondary analysis, tertiary analysis, diagnostic analysis, merging all images in the first processing result, displaying in the interaction unit, performing cell segmentation according to the interested region, analyzing the segmented cells, and generating information including the number of cells corresponding to the markers and the positive probability.

[0281] Specifically, the processor is further configured to control the interaction unit to feed back the second processing result of the user, and the second processing result includes at least one of secondary analysis results, tertiary analysis results, quantitative analysis, phenotype analysis, spatial analysis, and diagnostic report.

[0282] The above-described technical features can be combined arbitrarily. Although all possible combinations of the technical features are not described, any combination of the technical features should be considered to be covered by the present specification, as long as there is no contradiction in such a combination.

[0283] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above-described embodiments are exemplary, and it is not construed that the present disclosure is limited to the above-described embodiments, and a person of ordinary skill in the art can make changes, modifications, replacements, and variations to the above-described embodiments within the scope of the present disclosure.

Claims

1. An assay system integrating multiple detection modes, characterized in that, The assay system comprises: a stage for carrying a sample to be detected; an optical system selected from at least one or more of a combination of a light source, a lens group, and an objective lens; a detection system for detecting optical signals; a fluidic system for providing reagents for a detection reaction; a control system for controlling switching of multiple detection modes, and / or controlling the optical system, the fluidic system, and the detection system to perform detection of the sample to be detected according to a predetermined procedure in a detection mode.

2. The assay system of claim 1, wherein, Further comprising an analysis system for receiving information from the detection system, and outputting a detection result after analyzing the detection information.

3. The assay system according to claim 1 or 2, characterized in that The detection system is selected from a camera, a photodiode, an optical sensor, and the like.

4. The assay system according to any one of claims 1 to 3, characterized in that, The multiple detection modes are selected from a sequencing mode, a tissue sample staining mode, a DNA or RNA in-situ detection mode, a cell or microorganism staining detection mode, and the like.

5. The assay system of claim 4, wherein: one or more stages for carrying the same or different samples to be detected; one or more sets of optical systems selected from at least one or more of a combination of a light source, a lens group, and an objective lens; one or more sets of detection systems for detecting optical signals; one or more sets of fluidic systems for providing reagents for a detection reaction; one or more sets of control systems for controlling switching of multiple detection modes, or simultaneously performing multiple detection modes on multiple samples to be detected.

6. The assay system of claim 5, wherein, The control system further comprises: a driving circuit coupled to the detection system to collect signal data generated by the sample to be detected, the driving circuit being selectively coupled to mode parameters corresponding to an operation mode of the detection system, the mode parameters including at least first mode control parameters corresponding to the sequencing mode, and second mode control parameters corresponding to the tissue sample staining mode; a mode switching circuit coupled to the driving circuit to control the driving circuit based on the sample to be detected, the mode switching circuit associating a first set of mode parameters to the driving circuit when the sample slide is a sequencing chip, and associating a second set of mode control parameters to the driving circuit when the sample slide is a tissue sample slide.

7. The assay system of claim 5, wherein The control system further comprises: a plurality of independent driving circuits, each coupled to the detection system to collect signal data generated by the sample to be detected, the plurality of driving circuits being independently selectively coupled to mode parameters corresponding to an operation mode of the detection system, the mode parameters including at least first mode control parameters corresponding to the sequencing mode, second mode control parameters corresponding to the tissue sample staining mode, third control parameters corresponding to the DNA or RNA in-situ detection mode, and fourth control parameters corresponding to the cell or microorganism analysis detection. a mode switching circuit coupled to the driving circuit to control the driving circuit based on the sample to be detected, wherein the mode switching circuit associates a first set of mode parameters to the driving circuit when the sample slide is a sequencing chip, and associates a second set of mode parameters to the driving circuit when the sample slide is a tissue sample slide.

8. The assay system of any one of claims 1 to 7, wherein, The optical system further comprises a bright field light source and an excitation light source.

9. The assay system of claim 1, wherein, The sample to be detected comprises a nucleic acid sequencing library, a biological tissue sample, a biological fluid sample, a microorganism sample, or a cell sample fixed on the sample slide.

10. The assay system of claim 9, wherein, The optical system comprises an objective lens; in the sequencing mode, the optical system scans the nucleic acid sequencing library through the objective lens and generates sequence information corresponding to the nucleic acid sequencing library; In the tissue sample staining mode, the optical system scans the biological tissue sample through the objective lens and generates an image corresponding to the biological tissue sample.

11. The assay system of claim 7, wherein, The optical system comprises an objective lens; in the DNA or RNA in situ detection mode, the optical system scans the biological sample through the objective lens and generates a DNA or RNA in situ image corresponding to the biological sample; in the cell or microorganism analysis mode, the optical system scans the cell or microorganism sample through the objective lens and generates image information corresponding to the detection target of the cell or microorganism sample.

12. A multi-mode detector, characterized by, The multi-mode detection instrument comprises the assay system of any one of claims 1-11.

13. A multi-detection mode assay method, characterized by, The assay method comprises the following steps: fixing the sample to be detected on a stage; selecting a detection mode by a control system, comprising: selecting a first detection mode, starting a first fluid system corresponding to the first detection mode, and controlling the sample to be detected to generate a first detectable optical signal by a first detection reaction; and / or selecting a second detection mode, starting a second fluid system corresponding to the second detection mode, and controlling the sample to be detected to generate a second detectable optical signal by a second detection reaction; detecting the first optical signal and / or the second optical signal and transmitting to an analysis system; and analyzing the first optical signal and / or the second optical signal to generate detection information and output a detection result.

14. The assay method according to claim 13, characterized by The first detection mode and the second detection mode are the same detection mode, or the first detection mode and the second detection mode are different detection modes.

15. The assay method according to claim 14, characterized in that, When the first detection mode and the second detection mode are different detection modes, the first detection reaction and the second detection reaction are different detection reactions.

16. The assay method according to any one of claims 13 to 15, characterized in that, The plurality of detection modes are selected from nucleic acid sequence determination mode, tissue sample staining mode, DNA or RNA in situ detection mode, cell or microorganism analysis mode, etc.

17. The assay method according to any one of claims 13 to 16, characterized in that, The plurality of detection modes can be parallel.

18. The assay method of claim 16, wherein the detection reaction corresponding to the nucleic acid sequence determination mode is a sequencing reaction, wherein the sequencing reaction is selected from a synthesis sequencing reaction, a ligation sequencing reaction, a single molecule sequencing reaction, etc. The detection reaction corresponding to the tissue sample staining mode is an affinity reaction, wherein the affinity reaction is selected from antigen-antibody, protein-aptamer, biotin-streptavidin, nucleotide conjugated antibody-fluorescent probe, etc. The detection reaction corresponding to the DNA or RNA in-situ detection mode is a probe capture reaction, wherein the probe capture reaction is selected from probe conjugated fluorescence, probe ligation, probe amplification, etc., and the DNA or RNA can be selected from one of in-situ nucleic acid samples in a tissue sample, liquid nucleic acid samples such as blood and body fluids, and in-situ nucleic acid samples in cells or microorganisms. The detection reaction corresponding to the cell or microorganism analysis mode is surface antigen detection, biomarker detection, positive cell proportion analysis, transcriptome or epigenetic analysis, etc.

19. The assay method according to any one of claims 13 to 18, characterized in that, The control system comprises at least one processor and an interaction unit, which are coupled with the stage and the fluid system, a first instruction is input, the first instruction comprising: initial information, the initial information comprising at least one of a sample type to be detected, a sequencing scheme, a staining scheme, a kit number, and confirmation information; based on the initial information, the fluid system is controlled to perform a first detection reaction; based on the detection reaction, a first detection result is generated; the interaction unit is controlled to feed back the first detection result.

20. The assay method according to any one of claims 13 to 19, characterized in that, The fixing of the sample to be detected on the stage comprises: installing a plurality of slides to a plurality of stages, further comprising: installing a first slide to the first stage; installing a second slide to the second stage; the first slide and the second slide are fixed with different sample types, or the first slide and the second slide are fixed with the same sample type.

21. The assay method of claim 20, wherein, An optical system and a detection system are used to collect optical signals, wherein the optical system comprises a first optical link and a second optical link, and the detection method comprises: adjusting the relative position between the optical system and the stage so that any one of the first slide and the second slide is aligned with any one of the first optical link and the second optical link.

22. A mode switching method of a multi-mode assay system, characterized by, The mode switching method comprises: identifying the type of sample to be detected; based on the type of sample to be detected, determining mode parameters selected to be coupled to a driving circuit; when the sample to be detected is a nucleic acid sequencing library, the mode switching circuit associates a first set of mode parameters to the driving circuit, and the driving circuit drives the optical module into a sequencing mode; when the sample to be detected is a biological tissue sample, a microorganism sample or a cell sample, the mode switching circuit associates a second set of mode control parameters to the driving circuit, and the driving circuit drives the optical module into a fluorescent staining mode or a third mode different from the fluorescent staining mode and the detection mode.

23. The handover method of claim 22, wherein, The third mode is a bright field imaging mode.

24. The handover method of claim 23, wherein, The optical system comprises an excitation light source and a bright field light source; in the sequencing mode or the fluorescent staining mode, the excitation light source excites the sample to be detected to generate a fluorescent signal; in the bright field imaging mode, the bright field light source irradiates the sample to be detected to generate a bright field signal.

Images