WO2025218675A1 - Système de détermination ayant de multiples modes de détection, procédé de détection et procédé de commutation de mode associé - Google Patents

Système de détermination ayant de multiples modes de détection, procédé de détection et procédé de commutation de mode associé

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Publication number
WO2025218675A1
WO2025218675A1 PCT/CN2025/089147 CN2025089147W WO2025218675A1 WO 2025218675 A1 WO2025218675 A1 WO 2025218675A1 CN 2025089147 W CN2025089147 W CN 2025089147W WO 2025218675 A1 WO2025218675 A1 WO 2025218675A1
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WO
WIPO (PCT)
Prior art keywords
mode
sample
detection
sequencing
optical
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/089147
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English (en)
Chinese (zh)
Inventor
杨梦�
王毅雄
谢艳利
廖健君
李萌
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MGI Tech Co Ltd
Original Assignee
MGI Tech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MGI Tech Co Ltd filed Critical MGI Tech Co Ltd
Priority to CN202580000521.3A priority Critical patent/CN120584178A/zh
Publication of WO2025218675A1 publication Critical patent/WO2025218675A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Definitions

  • the present disclosure relates to the field of bioanalysis technology, and in particular to a measurement system with multiple detection modes, a detection method, and a mode switching method thereof.
  • Microscopy is a key technology in the life sciences, and all relevant detection technologies in this field use microscopes as a key means of observing biomacromolecules.
  • current microscopy equipment is limited in functionality and cannot perform complex, multifunctional detection tasks such as sequencing and multiplex staining and imaging on the same platform. This consumes significant manpower and material resources, and increases equipment costs. Therefore, a device that can achieve multifunctional detection at a lower cost is urgently needed.
  • the present disclosure addresses the drawbacks of the prior art and the above-mentioned problems, and provides a measurement system integrating multiple detection modes.
  • a first aspect of the present disclosure provides a measurement system integrating multiple detection modes, the system comprising:
  • a carrier the carrier being used to carry a sample to be tested
  • optical system wherein the optical system is at least selected from a combination of a light source, a lens group, and an objective lens;
  • a fluid system for providing a plurality of reagents for a detection reaction
  • a control system is used to control the switching of multiple detection modes, and/or to control the optical system, fluid system and detection system according to a predetermined program in one detection mode to complete the detection of the sample to be detected.
  • the measurement system further comprises an analysis system, which is used to receive information from the detection system, analyze the detection information, and output a detection result.
  • the detection system is selected from a camera, a photodiode, an optical sensor, and the like.
  • the multiple detection modes are selected from nucleic acid sequencing mode, tissue sample staining mode, DNA or RNA in situ detection mode, cell or microbial analysis mode, etc.
  • Another aspect of the present disclosure provides an assay system integrating multiple detection modes, wherein the assay system comprises:
  • One or more carriers each carrier being used to carry the same or different samples to be tested;
  • One or more optical systems wherein the optical systems are at least selected from a combination of a light source and a lens group;
  • one or more detection systems for detecting optical signals one or more detection systems for detecting optical signals
  • One or more fluid systems for providing a plurality of reagents for a detection reaction ;
  • control system is used to control the switching of multiple detection modes or to detect multiple samples to be detected in parallel using multiple detection modes.
  • 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 operating mode of the detection system, the mode parameters including at least a first mode control parameter corresponding to the sequencing mode and a second mode control parameter corresponding to the tissue sample dyeing mode;
  • a mode switching circuit is coupled to the driving circuit to control the driving circuit based on the sample to be detected: when the sample carrier is a sequencing chip, the mode switching circuit associates a first set of mode parameters to the driving circuit, and when the sample carrier is a tissue sample carrier, the mode switching circuit associates a second set of mode control parameters to the driving circuit.
  • control system further comprises:
  • 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, the plurality of driving circuits being independently selectable to be coupled to mode parameters corresponding to the operating mode of the detection system, the mode parameters 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 mode control parameter corresponding to the DNA or RNA in situ detection mode, and a fourth mode control parameter corresponding to the cell or microorganism analysis mode; and
  • multiple mode switching circuits are coupled to the driving circuit to control the driving circuit based on the sample to be detected: when the sample carrier is a sequencing chip, the mode switching circuit associates a first set of mode parameters to the driving circuit, and when the sample carrier is a tissue sample carrier, the mode switching circuit associates a second set of mode control parameters to the driving circuit.
  • the optical system further comprises a bright field light source and an excitation light source.
  • the sample to be detected includes a nucleic acid sequencing library, a biological tissue sample, a biological fluid sample, a microbial sample or a cell sample fixed on the sample slide.
  • 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.
  • 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.
  • Another aspect of the present disclosure discloses a method for measuring multiple detection modes, characterized in that the method comprises the following steps:
  • the control system selects the detection mode, including:
  • Selecting a first detection mode activating a first fluid system corresponding to the first detection mode, and controlling the sample to be detected to undergo a first detection reaction to generate a detectable first optical signal; and/or selecting a second detection mode, activating a second fluid system identical to the second detection mode, and controlling the sample to be detected to undergo a second detection reaction to generate a detectable second optical signal;
  • the first optical signal and/or the second optical signal are analyzed to generate detection information and output a detection result.
  • 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.
  • the first detection reaction and the second detection reaction are different detection reactions.
  • the multiple detection modes are selected from nucleic acid sequence determination mode, tissue sample staining mode, DNA or RNA in situ detection mode, cell or microbial analysis mode, etc.
  • the multiple detection modes can be performed in parallel.
  • the detection reaction corresponding to the sequencing 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 selected from affinity reactions, wherein the affinity reactions are selected from antigen-antibody, protein-aptamer, biotin-streptavidin, nucleotide-coupled antibody-fluorescent probe, etc.;
  • the detection reaction corresponding to the DNA or RNA in situ detection mode is selected from a probe capture reaction, wherein the probe capture reaction is selected from probe-coupled fluorescence, probe ligation, probe amplification, etc.;
  • 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.
  • control system includes at least one processor and an interaction unit, wherein the processor and the interaction unit are coupled to the carrier and the fluid system.
  • Input a first instruction, the first instruction including: initial information, the initial information including at least one of the type of sample to be tested, sequencing scheme, staining scheme, kit number, and confirmation information;
  • fixing the sample to be detected on the carrier includes: mounting a plurality of slides on a plurality of carriers includes:
  • the first carrier plate and the second carrier plate are fixed with different sample types, or the first carrier plate and the second carrier plate are fixed with the same sample type.
  • an optical system and a detection system are used to collect optical signals, wherein the optical system includes a first optical link and a second optical link, and the determination method includes:
  • the relative position between the optical system and the stage is adjusted to align either the first carrier or the second carrier with either the first optical link or the second optical link.
  • Another aspect of the present disclosure discloses a mode switching method for a system for measuring multiple detection modes, wherein the mode switching method comprises:
  • the mode switching circuit associates the first set of mode parameters with the driving circuit, and the driving circuit drives the optical module to enter the sequencing mode;
  • the mode switching circuit associates the second set of mode control parameters with the driving circuit, and the driving circuit drives the optical module to enter the fluorescence dyeing mode or a third mode different from the fluorescence dyeing mode and the measurement mode.
  • the optical system includes an excitation light source and a bright field light source;
  • the excitation light source excites the sample to be detected to generate a fluorescent signal
  • the bright field light source illuminates the sample to be detected to generate a bright field signal.
  • the assay system with multiple detection modes includes a control module, which is programmable and configured to control the fluid path module to introduce or discharge reagents into or out of the sample chamber, and control the imaging module to image the sample to be detected.
  • the system further includes an identification circuit coupled to the mode switching circuit having an interface for identifying the sample slide type and/or the operating mode.
  • the interface includes a user input port for receiving input of slide type information and/or the operating mode.
  • the sample slide has an identifiable type identification; and the interface includes a scanning mechanism for identifying the type of the sample slide based on the type identification.
  • the sample to be detected includes a nucleic acid sequencing library, a biological tissue sample, a biological fluid sample, an environmental sample, a microbial sample, or a cell sample fixed on the sample slide.
  • the optical module includes 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, microbial sample or cell sample.
  • the optical module includes an image sensor and an image processing unit operably connected to the image sensor, and the image processing unit is called by the second mode control parameter to receive multiple images corresponding to multiple FOVs generated by the image sensor and integrate and stitch the multiple images.
  • the optical module includes a first optical link and a second optical link that are independent of each other, the first optical link includes a first objective lens, the second optical link includes a second objective lens, and when the first optical link is in the sequencing mode, the second optical link is in the same or different mode as the first optical link.
  • the optical module also has a third operating mode different from the sequencing mode and the fluorescent staining mode, and the optical module also includes 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 operating mode at the same time.
  • the sample stage includes at least two detection positions for receiving a plurality of the sample slides.
  • the assay system for multiple detection modes includes a fluidic system arranged in a one-to-one correspondence with the detection sites.
  • the present disclosure further provides a detection method of a measurement system with multiple detection modes, comprising:
  • the mode switching circuit determines a mode parameter that is selectively coupled to the driving circuit, wherein the mode parameter corresponds to an operation mode of the optical module and includes at least a first mode control parameter corresponding to the sequencing mode and a second mode control parameter corresponding to the fluorescence staining mode;
  • the driving circuit applies the mode parameters determined by the mode switching circuit to the optical module to enable the optical module to switch between at least the sequencing mode and the fluorescence staining mode.
  • the detection method before or after installing the first slide to the first detection position of the sample stage, the detection method further includes: loading a first sample to be detected onto the first slide, wherein the first sample to be detected includes a nucleic acid sequencing library, a biological tissue sample, a microbial sample, or a cell sample.
  • the detection method before or after installing the second slide to the second detection position of the sample stage, the detection method further includes: loading a second sample to be detected onto the second slide, wherein the second sample to be detected includes a nucleic acid sequencing library, a biological tissue sample, a microbial sample or a cell sample.
  • the first sample to be tested is different from the second sample to be tested.
  • the third operating mode is a bright field imaging mode.
  • the technical solution disclosed in the present invention realizes multiple automated detection functions, has simple operation steps, more effectively utilizes resources, and saves equipment costs.
  • FIG1 is a simplified schematic diagram of a measurement system for multiple detection modes provided by an embodiment of the present disclosure
  • FIG2 is a schematic diagram showing the relationship between the optical module and the sample stage of the measurement system with multiple detection modes according to an embodiment of the present disclosure
  • FIG3 is a schematic diagram showing the relationship between the optical module and the controller of the measurement system with multiple detection modes according to an embodiment of the present disclosure
  • FIG4 is a partial schematic diagram of an optical module of a measurement system for multiple detection modes provided by an embodiment of the present disclosure
  • FIG5A is another schematic diagram of a measurement system with multiple detection modes provided by an embodiment of the present disclosure.
  • FIG5B is a schematic diagram of the optical path of a measurement system with multiple detection modes in a fluorescence staining mode according to an embodiment of the present disclosure
  • FIG5C is a schematic diagram of a detection system with multiple detection modes in a sequencing mode according to an embodiment of the present disclosure
  • FIG6 is a schematic flow chart of a detection method of a measurement system with multiple detection modes provided by an embodiment of the present disclosure
  • FIG7 is a flow chart of a method for switching modes of a measurement system with multiple detection modes provided by an embodiment of the present disclosure.
  • FIG8 shows a schematic diagram of the specific structure of a multifunctional biochemical detection system according to an exemplary embodiment of the present disclosure.
  • FIGS. 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.
  • 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;
  • Figure 10D shows the sample slide assembly being completed by covering the cover glass with the frame.
  • 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.
  • 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.
  • 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.
  • FIG14 is a schematic diagram of the structure of a computer terminal implementing software selectability in an exemplary embodiment of the present disclosure.
  • 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.
  • FIG16 is a flow chart of a multi-mode detection result analysis module in one embodiment of the present disclosure.
  • FIG17 shows the results of detecting single-cell transcriptomes in one embodiment of the present disclosure.
  • first and second are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as “first” or “second” may explicitly or implicitly include one or more of the features. Furthermore, in the description of this disclosure, unless otherwise specified, “plurality” means two or more.
  • first feature when a first feature is “above” or “below” a second feature, it may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Furthermore, when a first feature is “above,” “above,” or “above” a second feature, it may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. When a first feature is “below,” “below,” or “below” a second feature, it may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.
  • a fluidic system is equivalent to a fluidic module
  • an optical module is equivalent to an optical device
  • a carrier is equivalent to a sample stage
  • a slide is equivalent to a chip, etc., all of which are similar concepts known to those skilled in the art.
  • Omics analysis refers to the application of a range of high-throughput, large-scale, multidimensional technologies to study the collection of diverse molecules within an organism and their interactions.
  • the term "omics” derives from concepts in biology such as the genome, proteome, transcriptome, and metabolome, with each "omics” representing the complete set of molecules of a specific type within an organism.
  • Genome generally refers to genomic information from a subject, which can be, for example, at least a portion or all of the subject's genetic information.
  • a genome can be encoded in DNA or RNA.
  • a genome can include coding regions that encode proteins as well as non-coding regions.
  • a genome can include the sequences of all chromosomes in an organism. For example, the human genome has a total of 46 chromosomes. All of these sequences together may constitute the human genome.
  • Cytomics refers to the comprehensive study of all components within a cell, including its structure, function, behavior, and interactions. Cytomics encompasses all levels, from individual cells to cell populations, aiming to reveal the complexity and diversity of cells.
  • Transcriptome The study of the collection of RNA molecules, particularly mRNA, that represent the expression of genes at a specific time and under specific conditions. Transcriptome analysis helps understand how genes are regulated and their roles in biological processes.
  • proteome refers to the comprehensive study of the expression, function, interaction, and dynamic changes of all proteins in an organism, cell, or tissue.
  • the goal of proteomics is to establish a complete protein catalog, including protein identification, quantification, modification, and functional analysis.
  • Metabolome studies the collection of all small molecule metabolites in an organism. Metabolome analysis can reveal changes in metabolic pathways and the roles of metabolites in biological processes.
  • Sample also known as sample, refers to biological samples of different sources.
  • a biological sample can be a nucleic acid sample, a cell sample, a microbial sample, or a protein sample.
  • a biological sample can be derived from another sample.
  • a sample can be a tissue sample, such as a biopsy, a core biopsy, a needle aspiration, or a fine needle aspiration.
  • a sample can be a fluid sample, such as a blood sample, a urine sample, or a saliva sample.
  • a sample can be a skin sample.
  • a sample can be a cheek swab.
  • a sample can be a plasma or serum sample.
  • a sample can be cell-free or acellular.
  • a cell-free sample can include extracellular polynucleotides.
  • Extracellular polynucleotides can be isolated from body samples, and body samples can be selected from blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears. It can also be a microbial sample such as a virus or bacteria. Different sources mean that biological samples can come from human, animal, plant, microbial community, environmental samples, and even space samples.
  • Slides are a common experimental tool in biological 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.
  • Slides can be made of different materials, such as glass, silicon, resin, polymer, metal, metal oxide, etc. They can also be differentiated according to their use. For example, they can include sequencing chips, closed or open sequencing chips, semiconductor chips, nanopore chips, microscope slides, tissue microarrays (TMAs), cell arrays, protein arrays, biochips, microarrays, flow cytometry slides, tissue sections, etc.
  • TMAs tissue microarrays
  • Sequencing is a new type of genetic testing technology that can analyze and determine the complete sequence of gene bases from blood or saliva, thereby predicting the possibility of suffering from various diseases, individual behavioral characteristics and behavioral rationality. Sequencing generally refers to methods and techniques for determining the nucleotide base sequence in one or more polynucleotides.
  • Polynucleotides can be, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (for example, single-stranded DNA). Sequencing can be performed by various currently available systems, such as but not limited to sequencing systems from MGI, Sequencing, Illumina, Pacific Biosciences, Oxford Nanopore or Life Technologies (Ion Torrent).
  • nucleic acid amplification for example, nucleic acid amplification, polymerase chain reaction (PCR) (for example, digital PCR, quantitative PCR or real-time PCR) or isothermal amplification can be used for sequencing.
  • PCR polymerase chain reaction
  • Such devices can provide a plurality of raw genetic data corresponding to the genetic information of a subject (for example, a human), as generated by the device from a sample provided by the subject.
  • a sequencer is generally a device used to determine the sequence of the genetic material of a sample. Sequencers can function in a variety of ways and based on a variety of technologies, including sequencing by primer extension using labeled or unlabeled nucleotides, such as sequencing-by-ligation or pyrosequencing, for example using Sanger dideoxy sequencing, nanopore sequencing, or any of the "NexGen” sequencing methods known in the art (e.g., using the sequencing platform of MGI, the ROCHE 454 sequencing platform, the ILLUMINATM SOLEXATM sequencing platform, the SOLIDTM sequencing platform of LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS, the SMRTTM sequencing platform of PACIFIC BIOSCIENCES, the POLLONATOR Polony sequencing platform, the COMPLETE GENOMICS sequencing platform, the INTELLIGENT BIOSYSTEMS sequencing platform, the HELICOS sequencing platform, or any other sequencer or system known in the art).
  • sequencing platform of MGI the ROCHE 454
  • Multiple staining imaging can be multiple immunofluorescence staining (mIF) technology. Through multiple rounds of staining cycles, mIF can achieve in situ multi-target staining of tissues or cells, thereby comprehensively studying cell composition, cell function and cell-to-cell interactions. This method not only improves the sensitivity of detection, but also allows for analysis of the spatial distribution of multiple biomarkers in the sample. It can also be multiple fluorescent protein immunoblotting (multiple fluorescent Western Blot), which uses different fluorescent markers to detect multiple target proteins in a protein sample. This method reduces background fluorescence and avoids signal leakage by optimizing the sample load and selecting appropriate fluorescent markers, thereby achieving simultaneous detection of multiple proteins. It can also be other methods such as multiple ion beam imaging technology (MIBI), which can quantify proteins in single cells or even subcellular structures.
  • MIBI multiple ion beam imaging technology
  • Iterative indirect immunofluorescence imaging (4i) is an imaging technique developed by Professor Lucas Pelkmans at the University of Zurich that can use fluorescence microscopy to visualize dozens of proteins in thin tissue sections with high resolution.
  • a workstation is a high-performance computer system designed to meet the needs of professional users or specific industries. It typically features powerful processing capabilities, high-quality graphics, and extensive scalability, enabling it to handle complex computing tasks, data analysis, graphics rendering, and engineering design.
  • a sample analyzer is a device used to analyze and evaluate biological, chemical, or physical samples. It can be at least one of an optical microscope, electron microscope, fluorescence microscope, UV-Vis spectrophotometer, infrared spectrometer, nuclear magnetic resonance spectrometer, liquid chromatograph (HPLC), gas chromatograph (GC), mass spectrometer (MS), flow cytometer, gene sequencer, mass spectrometer, biochemical analyzer, cell counter, thermal cycler, electrophoresis equipment, biosafety cabinet, or lyophilizer.
  • an optical microscope electron microscope, fluorescence microscope, UV-Vis spectrophotometer, infrared spectrometer, nuclear magnetic resonance spectrometer, liquid chromatograph (HPLC), gas chromatograph (GC), mass spectrometer (MS), flow cytometer, gene sequencer, mass spectrometer, biochemical analyzer, cell counter, thermal cycler, electrophoresis equipment, biosafety cabinet, or lyophilizer.
  • the embodiments disclosed in the present disclosure provide a measurement system for multiple detection modes including an optical scanning device and related methods.
  • the measurement system for multiple detection modes is particularly used for biochemical sample detection, and includes a sample stage for holding a sample slide and an optical module for scanning the sample to be detected.
  • the optical module has a variable operating mode, and its operating mode is related to the type of sample to be detected. Different operating modes of the optical module involve calling different mode control parameters.
  • the optical module and the sample stage can be arranged to move relative to each other, and the operations of the optical module, such as the liquid circuit part that supplies fluid to the slide, the motion control module that controls the relative movement between the optical module and the sample stage, etc., which cooperate with the optical module, may also be adaptively adjusted in different operating modes of the optical module, such as the timing adjustment of the liquid supply and discharge speed of the liquid circuit part, the change of the driving parameters of the motion control part, etc.
  • the parameter groups corresponding to the different operating modes of the optical module can be predetermined and stored in the memory, for example, the optical module has any combination of a first operating mode (e.g., sequencing mode), a second operating mode (e.g., fluorescence staining mode), and a third operating mode (e.g., bright field imaging mode).
  • a first operating mode e.g., sequencing mode
  • a second operating mode e.g., fluorescence staining mode
  • a third operating mode e.g., bright field imaging mode
  • the first mode control parameters corresponding to the sequencing mode, the second mode control parameters corresponding to the fluorescence staining mode, and the third mode control parameters corresponding to the bright field imaging mode are pre-stored in the memory for selective call during actual operation.
  • the first mode control parameters include laser power parameters, real-time focus parameters, slide motion control parameters, fluid timing parameters, etc.
  • the real-time focus parameters include, for example, the real-time focus module SNR parameters of the sequencing chip supported by the adaptive algorithm.
  • the second mode control parameters include laser power parameters (e.g., 150mW and 300mW laser power), focus parameters (e.g., chip real-time focus SNR parameters based on tissue autofluorescence), and fluid timing parameters corresponding to the second mode (e.g., corresponding to immunofluorescence staining fluid timing), as well as splicing parameters, etc., which are different from the first mode control parameters.
  • the third mode control parameters include, for example, laser power parameters for 100mW and 200mW lasers, adaptive focus parameters for image scoring based on cell/microorganism fluorescence intensity, and image sensor parameters corresponding to cell/microorganism fluorescence. These control parameters are selectively invoked based on the type of sample being tested on the sample slide. Therefore, the system can dynamically invoke different mode control parameters based on changes in the sample being tested, switching the operating mode of the optical module in real time, and enabling multiple biochemical tests on biological samples without the need for additional equipment.
  • a sample slide 120 is used to carry the sample to be detected, and the sample slide 120 can be positioned on a sample stage 110, which is located below the objective lens of the optical module 110.
  • the sample slide can be fixed by vacuum adsorption or the like.
  • the optical module 100 and the sample stage 110 are relatively movably arranged.
  • the optical module 100 has at least a sequencing mode and a fluorescent staining mode. It should be understood that the optical module 100 can also have a third operating mode different from the sequencing mode and the fluorescent staining mode, such as a bright field imaging mode.
  • the operating 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 a nucleic acid sequencing library, a biological tissue sample, a microbial sample or a cell sample, etc.
  • the optical module 100 scans the nucleic acid sequencing library via the objective lens and generates sequence information corresponding to the nucleic acid sequencing library.
  • the optical module 100 scans the biological tissue sample via the objective lens and outputs an image corresponding to the biological tissue sample, microbial sample, or cell sample.
  • the optical module 100 captures and outputs a brightfield image of the sample to be tested.
  • the optical module 100 includes an image sensor and an image processing unit operably connected to the image sensor.
  • the image processing unit receives multiple images corresponding to multiple FOVs generated by the image sensor and integrates and stitches the multiple images.
  • the optical module 100 may include a first optical link 100a and a second optical link 100b that are independent of each other, and the sample stage 110 correspondingly includes at least two detection positions for receiving multiple sample slides.
  • the first optical link 100a includes a first objective lens
  • the second optical link 100b includes a second objective lens.
  • the first optical link is in the sequencing mode
  • the second optical link is in the same or different mode as the first optical link.
  • the operating mode control of the two optical links can be independent of each other.
  • the optical module 100 may also include 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 the third operating mode at the same time.
  • the optical module's light source module includes multiple light source elements, such as an excitation light source L1 and a brightfield light source L2. These light sources are selectively connected to, for example, a multiplexer MUX.
  • the multiplexer MUX is controlled by a controller 200 and selectively outputs light signals matching the operating mode of the optical module 100 to the sample to be tested. Illuminated by the light signal, the sample to be tested generates a corresponding detectable light signal. For example, in sequencing mode or fluorescence staining mode, the sample is excited by laser light to generate a fluorescence signal; in brightfield imaging mode, the sample receives brightfield light to generate a brightfield signal. This detectable light signal is transmitted through an objective lens and captured by an associated optical sensor.
  • the optical sensor In sequencing mode, the optical sensor is connected to a corresponding base recognition device, which ultimately converts the detectable light signal into sequence information and outputs it.
  • the optical sensor In fluorescence staining mode or brightfield imaging mode, the optical sensor is connected to an image processing device, which outputs the detectable signal as an image.
  • the optical module includes an image sensor and an image processing unit operably connected to the image sensor. The image processing unit receives multiple images corresponding to multiple FOVs generated by the image sensor in the fluorescence staining mode and integrates and stitches these images.
  • the sample stage 110 is movable relative to the objective lens to facilitate scanning of the objective lens at multiple detection locations of the sample to be detected.
  • Phase shift between the sample stage 110 and the objective lens can be achieved by moving the sample stage itself, the objective lens, the entire optical table, or any combination thereof.
  • the controller 200 includes a driver 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 from the sample to be detected.
  • the driver circuit 210 is selectively coupled to mode parameters corresponding to the operating mode of the optical module 100.
  • the mode parameters include at least a first mode control parameter corresponding to the sequencing mode and a second mode control parameter corresponding to the fluorescence staining mode.
  • the mode switching circuit 220 is coupled to the driver circuit 210 to control the driver circuit based on the sample to be detected.
  • the mode switching circuit 220 associates a first set of mode parameters with the driver circuit 210, and when the sample slide is a tissue slide, the mode switching circuit 220 associates a second set of mode control parameters with the driver circuit 210. After the type of the sample to be detected is determined, the mode switching circuit 220 selects the mode control parameters corresponding to the detected sample type. Exemplarily, the mode control parameters may be stored in a memory or other similar storage device.
  • the driver circuit 210 calls the mode switching circuit 220 to match the mode control parameters and loads them into the corresponding circuits of the optical module 100.
  • the host computer control software integrates the startup parameters corresponding to different modes. When the first set of mode parameters is activated, the host computer can automatically switch to/start the sequencing mode; when the second set of mode parameters is activated, the host computer can automatically switch to/start the fluorescence staining mode.
  • the controller 200 further includes an identification circuit coupled to the mode switching circuit 220, and in particular, includes an interface for identifying the sample slide type and/or the operating mode.
  • the interface for example, includes a user input port for receiving input slide type information and/or the operating mode.
  • the sample slide has a recognizable type identifier, such as a barcode or QR code; the interface includes a scanning mechanism (e.g., a barcode scanner) for identifying the type of the sample slide based on the type identifier and transmitting the scanned information to the mode switching circuit 220.
  • a scanning mechanism e.g., a barcode scanner
  • the present disclosure discloses a detection method for the aforementioned multi-detection mode measurement system.
  • a slide 120 is mounted on a sample stage 110.
  • the multi-detection mode measurement system is configured with multiple sample stages and optical links.
  • the first slide is mounted to the first detection position of the sample stage; the second slide is mounted to the second detection position of the sample stage.
  • the detection method further includes: loading a first sample to be detected onto the first slide, the first sample to be detected includes a nucleic acid sequencing library, a biological tissue sample, a microbial sample or a cell sample.
  • the detection method further includes: loading a second sample to be detected onto the second slide, the second sample to be detected includes a nucleic acid sequencing library, a biological tissue sample, a microbial sample or a cell sample.
  • the types of samples to be detected on the first slide and the second slide may be different or the same.
  • the optical module includes a first optical link and a second optical link
  • the detection method includes: adjusting the relative position between the optical module and the sample stage so that either the first slide or the second slide is aligned with either the first optical link or the second optical link.
  • the mode switching circuit determines the mode parameters selected for coupling to the driver circuit.
  • the mode parameters correspond to the operating mode of the optical module and include at least a first mode control parameter corresponding to the sequencing mode and a second mode control mode parameter corresponding to the fluorescence staining mode.
  • the mode parameters may further include, for example, a third mode control mode parameter corresponding to a brightfield imaging mode.
  • step S13 the driving circuit applies the mode parameters determined by the mode switching circuit to the optical module so that the optical module switches at least between the sequencing mode and the fluorescence staining mode.
  • an embodiment of the present disclosure further provides a mode switching method for the above-mentioned measurement system with multiple detection modes.
  • the mode switching method includes: S21, identifying the type of sample to be detected, especially when the slide at the detection site on the sample stage is replaced.
  • the measurement system with multiple detection modes is equipped with a sensor that monitors the status of the slide at the detection site in real time, or monitors the status through scanning imaging and machine vision.
  • the identification circuit is driven to execute the above-mentioned step S21.
  • steps S231 - 232 determine the mode parameters selected to be coupled to the driving circuit.
  • Step S24 when the sample to be detected is a nucleic acid sequencing library, the mode switching circuit associates the first set of mode parameters with the driving circuit, and the driving circuit drives the optical module to enter the sequencing mode; when the sample to be detected is a biological tissue sample, a microbial sample or a cell sample, the mode switching circuit associates the second set of mode control parameters with the driving circuit, and the driving circuit drives the optical module to enter the fluorescence staining mode or a third operating mode different from the fluorescence staining mode and the sequencing mode.
  • the third operating mode is the bright field imaging mode.
  • the real-time change of the sample type can be detected by the above-mentioned recognition circuit, and the new operating mode parameters are selected and called into the corresponding control loop of the optical module.
  • the calling of the operating mode parameters can occur in real time (but may still depend on system delays or other delays), so that the operating mode of the optical system can be changed dynamically.
  • a sample slide in a measurement system with multiple detection modes according to an embodiment of the present disclosure, includes a sample cavity for placing a sample to be detected, wherein the sample cavity is a sealed cavity provided with an exchange hole, and the exchange hole is used to introduce fluid into the sample cavity or discharge fluid from the sample cavity.
  • a liquid path module for introducing fluid into or discharging fluid from the sample cavity includes a reagent cavity and a pipeline connecting the reagent cavity with the sample cavity; and an imaging module for imaging the sample to be detected.
  • the sample cavity can be a detachable component to facilitate loading of the sample to be tested therein, and it can be loaded before use.
  • the sample cavity is a detachable sample slide.
  • the exchange hole includes a liquid inlet and a liquid outlet, and there can be one or more liquid inlet and liquid outlet, and more than one can be selected.
  • the fluid When the fluid is introduced, the fluid enters the sample cavity through the liquid inlet, and the air is discharged from the sample cavity through the liquid outlet; when the fluid is discharged, the fluid is discharged from the sample cavity through the liquid outlet, and a new fluid (liquid or gas) replaces the original fluid and enters the sample cavity.
  • the entry and discharge of the fluid can be carried out by a pump, such as a micro pump.
  • each of the multiple reagent chambers is connected to the sample chamber via a respective pipeline, and each pipeline is provided with a selection valve for introducing different reagents into the sample chamber as needed.
  • a control module may be used to control the sequential introduction of multiple reagents from the multiple reagent chambers into the sample chamber and the discharge of the multiple reagents from the sample chamber.
  • a temperature control module can be used to control the temperature of the fluids in the sample chamber and the reagent chamber, with the goal of ensuring that the incubation of the sample to be tested is carried out at a set temperature. Any method can be used to maintain the temperature of the fluids in the sample chamber and the reagent chamber, such as using an electric heater and a temperature-sensitive sensor to adjust the on and off of the electric heater. It should be understood that the temperature control parameters of the temperature control module may vary depending on the sample to be tested.
  • the optical path of the optical module 100 is schematically shown in Figure 5B .
  • the optical module 100 includes an objective lens 1001, an eyepiece 1006, fluorescence imaging cameras 1007-1008, and spectroscopic/filtering components.
  • the objective lens 1001 is positioned opposite the sample slide 120 to collect optical signals from the sample slide 120. Fluorescent signals within the optical signals are transmitted by the filter assembly 1003.
  • the light source module includes an epi-illumination source L2a and a transillumination source L2b, which can be selectively activated by the user according to actual needs.
  • the epi-illumination light source L2a is, for example, an epi-illumination LED light source.
  • the light passes through the filter assembly 1003 and enters the objective lens 1001, uniformly illuminating the tissue slice area of the sample slide 120 within the field of view of the objective lens 1001.
  • the fluorescent material in the sample slide 120 is stimulated to emit fluorescence.
  • the objective lens 1001 collects the fluorescence and collimates the output.
  • the fluorescence After being filtered by the filter assembly 1003, the fluorescence enters the tube lens, which magnifies the fluorescence and forms an image on the eyepiece 1006 or the sensor chip of the fluorescence imaging camera 1007-1008.
  • a binocular beam splitter is also provided between the eyepiece 1006 and the filter assembly 1003.
  • the sample slide 120 moves linearly at a constant speed, and the objective lens 1001 scans the imaging area of the biological tissue slice.
  • an additional focus detector can be provided to monitor the defocus of the biological tissue slice in real time.
  • a focusing mechanism is used to ensure that the tissue slice remains within the allowable defocus range.
  • the epi-illumination light source L2a can selectively emit epi-fluorescence and epi-brightfield light. Therefore, the optical path shown in FIG5B is also suitable for brightfield imaging mode.
  • any of cameras 1007-1008 must be switched to a color camera, and filter block 1003 must be switched to a brightfield filter block.
  • brightfield imaging mode can also utilize transillumination: the transillumination LED light source L2b emits illumination light, which is collimated and then passes through a reflector before entering a condenser.
  • the condenser focuses the light onto the sample in the sample carrier 120 to provide strong illumination.
  • the brightfield light signal from the sample carrier 120 is magnified by objective lens 1001 and other devices, and an image is projected onto the eyepiece 1006 or the sensor chip of cameras 1007-1008 (in this case, any of cameras 1007-1008 can be replaced with a color camera).
  • the sample slide 120 includes multiple sample receiving sites 1201.
  • a DNA sample is placed at a sample site 1201, where it emits a fluorescent signal.
  • a light source (not shown) in the optical system emits a light signal toward the sample site, stimulating the sample to produce a fluorescent signal.
  • a light-transmitting layer (equivalent to a microlens) corresponding to each sample site transmits the fluorescent signal from the sample site 1201 to a corresponding photosensor 1005.
  • the photosensor 1005 collects the fluorescent signal and generates an analog electrical signal.
  • a control circuit electrically connected to the photosensor 1005 then processes the analog electrical signal to determine the type and/or sequence of bases contained in the sample.
  • Figure 8 shows a schematic diagram of a multi-detection mode measurement system according to an exemplary embodiment of the present disclosure.
  • the multi-detection mode measurement system shown in Figure 8 can include both sequencing and fluorescence staining modes, further expanding the functionality of a sequencer and achieving multifunctionality.
  • the multi-detection mode measurement system includes a slide for placing samples to be detected, an optical module for optically scanning the samples to be detected, a fluidic module for introducing and removing liquid reagents from the samples to be imaged, a temperature control module for controlling the temperature of the fluids in the sample and reagent chambers, and a control module for controlling the rectification system.
  • the slide's sample chamber can be a sealed cavity with an exchange port for introducing or removing fluid from the sample chamber.
  • the exchange port includes an inlet and an outlet.
  • the sample chamber is a removable component, for example, a removable sample slide can be used as the sample chamber.
  • the optical module optionally includes a light source module, an optical detector, motion control, and autofocus mechanisms to implement optical scanning. Scanning a sample of a known focal length can ensure a clear image of at least one layer by capturing images from several layers above and several layers below.
  • the fluidic module includes a reagent chamber and pipelines connecting the reagent chamber to the sample chamber. Multiple reagent chambers can be provided to accommodate various reagents, each connected to the sample chamber via a pipeline.
  • a selector valve can be provided on the pipeline to selectively connect the reagent chamber to the sample chamber.
  • Supplying multiple reagents sequentially to the sample chamber allows for multiple incubations of multiple samples to be tested. Washing can be performed between incubations with different reagents, optionally using water or buffer. Incubation and washing can be programmed.
  • the control module automatically selects and drives fluids, allowing multiple reagents to be sequentially introduced into and discharged from the sample chamber from the multiple reagent chambers.
  • a temperature control module can separately control the temperatures of the sample chamber and reagent chamber, allowing them to be the same or different.
  • the temperatures of the multiple reagent chambers can be controlled separately, allowing the temperatures of different reagent chambers to be the same or different.
  • Automated temperature control can be achieved through the control module.
  • the temperature control module can employ a PID control method to adjust the temperature. Temperature changes can be achieved using Peltier elements built into the sample stage.
  • the control module can be run by a software system and executed by a main control computer, and is responsible for controlling the operation of the measurement system of various detection modes.
  • the control module is used to control the liquid circuit module to exchange reagents on the sample slide, and to control the microscope imaging module to image the sample slide, so that the incubation and imaging of the sample slide are carried out in the required order.
  • the editable configuration file allows the settings of the control module to be modified, and the customizable protocol file formulates the fluid processing, temperature setting and imaging steps in sequence.
  • the position of the sample chamber can be moved by a mobile platform (for example, by an x, y, z three-axis mobile platform) to accurately position and move the sample on the chip to ensure the superposition of multiple images and image stitching.
  • the scheme using the disclosed system combines the automation of microscopy and sample processing to visualize tens of thousands of features in millions of pixels of the sample.
  • the present disclosure can realize a simple and effective multiplexed, sensitive in situ protein detection method with programmable signal amplification.
  • the present disclosure can enable higher throughput imaging analysis from super-resolution studies to centimeter-scale tissue mapping work. These omics methods require precise control of temperature, reagent application and image acquisition parameters during iterative chemistry and imaging cycles of days or weeks. Automated execution of these methods enables robust and reproducible data generation.
  • the sample slide can be a sequencing slide for securing a nucleic acid detection library, or it can be another slide for supporting, for example, a biological tissue sample.
  • the sample slide is secured to a sample stage.
  • Figures 9A-9B show schematic diagrams of a sample slide according to an exemplary embodiment of the present disclosure.
  • Figure 9A shows a sample slide assembly diagram
  • Figure 9B shows an exploded view of the sample slide assembly.
  • Figures 10A-10D show schematic diagrams of attaching tissue sections to the sample slide of an exemplary embodiment. This secures the tissue sections to facilitate subsequent incubation and prevents them from moving in the liquid during incubation and washing.
  • Figure 10A shows the process of attaching the tissue sections to the slide
  • Figure 10B shows the state after the tissue sections have been attached to the slide
  • Figure 10C shows the attachment of double-sided tape to the slide
  • Figure 10D shows the completion of the sample slide assembly by placing a cover glass on the frame.
  • the sample slide is secured to a chip operating table.
  • the present disclosure is suitable for double-layer slide flow channel designs for tissue sections in the form of paraffin tissue sections, frozen tissue sections, and the like.
  • the tissue slices are transferred to a slide and fixed to the slide.
  • FIG. 11 shows a schematic diagram of the sample slide adapter fixing fixture.
  • the chip is placed on the chip fixing position of the base, and pressure is applied downward to the pressing block to compress the chip.
  • the base is made of engineering plastic and has a certain hardness; the pressing block is made of silicone, and forms a pressing effect on the chip with the base, but at the same time will not damage the chip.
  • the measurement system with multiple detection modes disclosed in the present invention integrates multi-mode detection schemes such as sequencing mode and fluorescent staining mode to form a multifunctional platform for staining, imaging and sequencing.
  • the measurement system with multiple detection modes can expand the application of sequencers, realize the integration of different applications, and improve the use value of the instrument.
  • the present invention transforms the sequencer into a multifunctional platform that is compatible with protein tissue staining and imaging in addition to gene sequencing functions.
  • the experimental design includes synchronous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity of the extended experiment.
  • this solution can realize the cyclic indirect immunofluorescence imaging (4i) function of complex multi-day continuous workflows without supervision.
  • the system disclosed in the present invention can perform highly automated 4i methods during the fluorescent staining imaging process, and is compatible with the vast majority of readily available antibodies on the market. After multiple cycles of staining, imaging and antibody elution, a highly multiplexed map of the cell types and pathological characteristics of animals or plants is finally constructed.
  • the scheme disclosed in the present invention 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.
  • the optical system is modified to realize a multiplexing scheme of more than multiple (for example, 6) labels per round through multispectral imaging.
  • multiplexing scheme of more than multiple (for example, 6) labels per round through multispectral imaging.
  • the disclosed scheme 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 disclosed multiplex biochemical detection is compatible with a variety of antibody fluorescence staining reagent schemes, including 4i based on fluorescently labeled secondary antibody amplification, TSA signal amplification, fluorescently labeled nucleotide-coupled antibody CODEX (co-detection through indEXing), etc. Among them:
  • Fluorescent labeling amplification technology Cyclic indirect immunofluorescence imaging (4i for short) is the first imaging technology that can provide a multiplexed perspective from tissue to organelle. In the same experiment, correlated multiplexed information at the tissue, cellular and subcellular levels can be obtained.
  • Immunofluorescence (IF) uses antibodies to visualize and locate proteins in biological samples. Standard IF can usually only label 2-3 proteins. 4i uses ready-made antibodies and conventional fluorescence microscopes. By repeatedly removing antibodies and hybridizing, it can visualize 10 times the number of proteins of IF.
  • TSA Teyramide Signal Amplification
  • HRP horseradish peroxidase
  • TSA is a tissue section multi-labeling solution based on tyramide signal amplification technology. It utilizes horseradish peroxidase (HRP) for high-density in situ labeling of target proteins or nucleic acids, enhancing detection sensitivity and significantly increasing the signal-to-noise ratio by over a thousand-fold. It can perform fluorescent multi-labeling of multiple markers on the same tissue section using primary antibodies from the same animal source. Its excellent signal amplification performance can boost signal intensity by 10-100 times, significantly improving detection sensitivity for weak signals and difficult-to-label proteins.
  • HRP horseradish peroxidase
  • the CODEX platform combines oligonucleotide labeling (barcode) and microfluidic automated staining technology to enable the detection and in-depth 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 each antibody with a specific oligonucleotide "barcode tag" (barcode), rather than directly labeling it with a fluorescent dye.
  • the fluorescent dye required for imaging is specifically bound to the oligonucleotide sequence complementary to the barcode, allowing CODEX to break the limitation of the number of visible spectrum fluorescence imaging channels and easily achieve the simultaneous detection and analysis of 50 or more protein indicators.
  • RNA and DNA targets complementing single-cell RNA sequencing analysis with functional information on protein expression, and will prove to have a wide range of potential applications, including tissue atlases, tumor and disease atlases, in situ single-cell validation of large-scale assays such as flow cytometry or mass cytometry, and CITE-Seq (single-cell sequencing of cell surface proteins and RNA simultaneously), as well as digital pathology and biomarker screening and discovery.
  • the disclosed method is also applicable to using DNA or RNA probes to detect the location of the complementary strand in bacteria or other eukaryotic cells.
  • RNA in situ nucleic acid hybridization also known as RNA in situ hybridization histochemistry or RNA in situ hybridization
  • RNA in situ hybridization is an in situ hybridization technique that uses probes such as cRNA or oligonucleotides to detect RNA expression in cells and tissues.
  • the basic principle is that, under conditions where the cell or tissue structure remains unchanged, labeled RNA nucleotide fragments are used to bind (hybridize) to corresponding gene fragments in the cells or tissues being tested, following the base pairing principle of nucleic acid hybridization.
  • the resulting hybrids undergo a colorimetric reaction, and the corresponding mRNA, rRNA, and tRNA molecules within the cells are visualized under an optical or electron microscope.
  • RNA in situ hybridization has expanded its application beyond DNA in situ hybridization. It is particularly effective in genetic analysis and diagnosis, enabling qualitative, localized, and quantitative analysis, making it a highly effective molecular pathology technique. It has also become a significant area of molecular biology for analyzing low-abundance and rare mRNA expression. Detection of nucleic acid targets can be achieved through probe hybridization, where oligonucleotide probes reversibly hybridize to the target nucleic acid, undergoing extension and ligation reactions.
  • oligonucleic acid probes can directly connect detectable labels, such as fluorescent labels, electrochemical labels, magnetic beads, nanoparticles, biotin, etc., for quantitative or detection of the oligonucleotide product connected.
  • detectable labels such as fluorescent labels, electrochemical labels, magnetic beads, nanoparticles, biotin, etc.
  • oligonucleic acid probes can also not connect detectable signals, and oligonucleic acid probes are specific to the target of interest, and produce connection products at each target site, can carry out the amplification reaction of signal amplification, produce multiple copies of connection products, utilize signal probe hybridization amplification products, thereby amplify detectable signals.
  • nucleic acid target is RNA target, and RNA target can be reverse transcribed to produce cDNA.Then cDNA can be reverse transcribed to produce cDNA.
  • a padlock probe can be contacted with a DNA target and hybridized to the DNA target.
  • the padlock probe can then be cyclized and amplified.
  • the amplified product or amplicon can be directly combined with a signal probe for in situ signal detection.
  • Figure 15 shows in situ mRNA detection data obtained using RNA in situ hybridization sequencing technology.
  • the signaling probe can be a sequence-specific oligonucleotide probe that is optically active when hybridized to a nucleic acid target or a derivative thereof (e.g., an amplification product).
  • the probe can be linked to any optically active (e.g., dye) described herein and can also include a quencher that can block the optical activity of the associated dye.
  • optically active e.g., dye
  • Non-limiting examples of probes that can be used as reporters include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.
  • Figure 12 shows a schematic diagram of the liquid circuit module of a measurement system for multiple detection modes according to an embodiment of the present disclosure.
  • the measurement system for multiple detection modes includes multiple groups of reagent chambers 1 for storing multiple reagents.
  • the reagent chamber 1 is connected to the sample slide 2 serving as the sample cavity through a pipeline, and a selection valve 3 is provided on the pipeline for selecting the reagents in different reagent chambers 1.
  • the sample slide 2 is also connected to the waste liquid barrel 5 through a pipeline for discharging the reagent from the sample slide 2.
  • An injection pump 4 is installed on the recovery line connected to the waste liquid barrel 5.
  • the injection pump 4 provides power, and the reagent is drawn out from the reagent chamber 1, enters the chip 2 through the precision fluid system, acts on the tissue in the chip 2, and is then drawn out from the chip 2 and finally enters the waste liquid barrel 5.
  • the microfluidic system can ensure rapid reaction between the target and the reagent and highly stable repeatability.
  • sample preparation may include the following steps: a) setting up a glass slide so that the sample is firmly bonded to the glass slide; b) after the sample is mounted, it can be pre-treated by traditional methods before being loaded on the machine; c) after being loaded on the machine, it can be heated by a temperature control system and antigen repair can be performed under strong acid or strong base conditions as needed.
  • the purpose of antigen repair is to fully expose the antigen cross-linked with paraffin so that the antibody can bind to the relevant antigen.
  • the acid, base, heat or pressure used is adjusted according to the specific antibody; d) using 0.5% TritonX-100 to incubate and punch the sample for 15 minutes.
  • the entire sample preparation process can be reduced from 300 minutes to about 180 minutes.
  • the sample can be pre-treated for multiple fluorescent staining as follows: a) Fluidity washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute); b) Blocking treatment with a blocking reagent (such as 10% BSA, 5% Goat Serum or 10% skim milk, etc.) for 30 minutes; c) Fluidity washing with PBS (time: 100 seconds, flow rate: 1200 microliters per minute); d) Add imaging buffer (Trolox, N-acetyl cysteine, DTT and other antioxidant reagent solutions); e) Perform a full-slice scan to obtain autofluorescence background for correction.
  • a blocking reagent such as 10% BSA, 5% Goat Serum or 10% skim milk, etc.
  • the method disclosed in the present invention can reduce the time to 5.3 hours or even shorter, while the traditional fluorescent staining process takes up to 8 hours or even longer. Moreover, most of the operations of the method disclosed in the present invention are completely automatically operated by the machine and do not require human participation.
  • Immunofluorescence detection is a secondary antibody amplification method:
  • imaging buffer (Trolox, N-acetyl cysteine, DTT and other antioxidant reagent solutions);
  • Antibody elution was performed using an antibody elution buffer (weakly acidic 2ME + SDS + TrisHCl solution, acidic glycine + SDS solution, or acidic glycine + urea + GC + TCEP solution) at a temperature controlled temperature (25-56 degrees Celsius);
  • Block the sample again with a blocking reagent such as 10% BSA, 5% Goat Serum, or 10% skim milk
  • a blocking reagent such as 10% BSA, 5% Goat Serum, or 10% skim milk
  • imaging buffer (Trolox, N-acetyl cysteine, DTT or other antioxidant reagent solution);
  • an antibody elution buffer weakly acidic 2ME + SDS + TrisHCl solution, acidic glycine + SDS solution, or acidic glycine + urea + GC + TCEP solution
  • a quencher 0.1 M sodium bicarbonate + 3% hydrogen peroxide, sodium borohydride, 24 mM sodium hydroxide + 4.5% hydrogen peroxide, etc.
  • viii Block the sample again with a blocking reagent (e.g., 10% BSA, 5% Goat Serum, or 10% skim milk) for 30 minutes.
  • a blocking reagent e.g., 10% BSA, 5% Goat Serum, or 10% skim milk
  • Immunofluorescence staining is nucleotide-coupled fluorescence:
  • nucleotide-conjugated primary antibody diluted in 1% BSA solution (1:10-1:400) and incubate at 37°C for 15-45 minutes.
  • the nucleotides coupled to the primary antibody can be amplified by RCA, PER (primer replacement method) and other amplification methods;
  • vii. Add the corresponding fluorescently labeled nucleotide duplex to a solution (PBS, TBS, etc.), add the sample, and incubate for 30 minutes. The temperature can be gradually lowered to enhance specificity.
  • Imaging buffer (Trolox, N-acetyl cysteine, DTT or other antioxidant reagent solution);
  • Double-stranded DNA can be eluted by high-temperature unwinding, degradation by double-stranded DNA degrading enzyme, unwinding and eluting by DNA helicase, etc. to elute the fluorescently labeled nucleotide duplex;
  • the present disclosure can add a reducing agent to the reagent to free the antibodies from cross-linking caused by photography, making them easier to elute.
  • the multi-mode measurement mode is to perform staining and photography mode, and then the instrument can switch to sequencing mode to sequence the prepared nucleic acid library.
  • the specific steps are as follows:
  • Figure 13 shows the results of multiple fluorescent staining of mouse brain sections captured using the multi-mode detector imaging platform. Green areas are stained with GFAP antibodies, and red areas are stained with fibronectin antibodies. Imaging parameters were: laser intensity: red—300mW, green—150mW; exposure time: 40ms.
  • the multi-mode detection instrument can perform mRNA in situ hybridization detection.
  • An example of a protocol based on mRNA in situ hybridization is given below.
  • the in situ detection method is mRNA in situ hybridization detection.
  • Figure 15 shows the results of multiple fluorescence staining of mouse tumor model tissue sections purchased from Wuhan Sevier, captured using a multi-mode detector imaging platform. Green areas represent Cd8 mRNA, and red areas represent Ki67 mRNA. Imaging settings: Laser intensity: Red—300mW, Green—150mW; Exposure time: 40ms.
  • the system and method disclosed in the present invention liberate manpower by realizing a highly automated staining + photography process that can be unattended; compared with the dye-shooting separation instruments in the prior art, the dye-shooting functions can be integrated into the instrument; the scheme disclosed in the present invention can accurately control the amount of reagents used, thereby generating stable data for multiple rounds of quantitative analysis; the scheme disclosed in the present invention supports multiple immunofluorescence staining of a single slice and is compatible with a variety of biochemical staining schemes; the scheme disclosed in the present invention can greatly optimize the reagent reaction time.
  • chip preparation may include the following steps: a) setting up a sequencing chip, fixing capture probes on the chip surface; b) loading a cell suspension onto the chip surface, fixing it and scanning and photographing it (fluorescence photography or bright field photography may be optional as needed); c) permeabilizing the cells so that the mRNA in the cells can pass through the cell membrane and be captured by the capture probes on the chip surface; d) performing reverse transcription and sequencing library construction on the chip surface; e) switching the multi-mode detector to sequencing mode, sequencing to obtain transcriptome data; f) integrating and analyzing the transcriptome data and cell photographic data to obtain single-cell transcriptome data.
  • the method disclosed herein is automatically operated by the multi-mode detector and does not require human participation.
  • chip preparation may include the following steps: a) setting up a sequencing chip, fixing capture probes on the chip surface; b) extracting the nuclei of cells or tissues; c) adding transposition complexes and incubating the nuclei for transposition reaction; d) loading the nucleus suspension onto the chip surface, fixing it and scanning and photographing it (fluorescence photography or bright field photography can be selected as needed); e) lysing the nuclei, and the genomic fragments interrupted by the transposition complex are captured by the capture probes on the chip surface; f) amplification and extension and sequencing library construction are performed on the chip surface; g) switching the multi-mode detector to sequencing mode, sequencing to obtain epigenetic group data; h) integrating and analyzing the epigenetic group data and the nucleus photograph data to obtain single-cell epigenetic group data.
  • the single-cell transcriptome data and epigenetic data can be integrated and
  • the cell or microbial analysis and detection result and sequencing result analysis process includes: S31, scanning the cell chip and taking a photo to obtain a scanned image; S32 obtaining sample nucleic acid sequence information in sequencing mode; S33, analyzing and integrating the sequencing data and cell images; S34, performing cluster analysis on the cell transcriptome data to obtain single-cell transcriptome data.
  • Fixation Load pre-cooled methanol and incubate at low temperature for 20 minutes to fix the cells on the chip surface, and scan and take pictures;
  • Permeabilization Dilute 0.01M HCl 10-fold to obtain the working solution concentration (working solution volume is 100 ⁇ L/chip), load onto the chip surface and incubate at 37°C for 0-30 minutes;
  • Reverse transcription The capture probes fixed on the chip surface hybridize with the mRNA in the cells and then perform reverse transcription (BGI Stereo-seq Transcriptome Kit, Cat. No. 101KT114) to obtain cDNA.
  • Figure 17 shows a partial capture of the chip image, showing a large number of single cells distributed across the chip. Circle one of the cells to see the average number of genes and UMIs captured for that cell.
  • Figure 14 is a computer system for implementing the present measurement system or method in one embodiment of the present disclosure.
  • the control system is implemented by a computer system, which is programmed or otherwise configured to use the method of the present disclosure to control the optical system, fluid system, detection system and analysis system to collaboratively process the sample to be tested according to a predetermined program.
  • 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, reacting 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 into a multi-mode measurement system or a computer system remotely located relative to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system includes a central processing unit (CPU, also referred to herein as a "processor” and “computer processor”), which can be a single-core or multi-core processor, or multiple processors for parallel processing.
  • the computer system also includes memory or storage location 310 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit (e.g., a hard disk), a communication interface (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices such as cache, other memory, data storage, and/or an electronic display adapter.
  • the memory, storage unit, interface, and peripheral devices communicate with the CPU via a communication bus (solid line), 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") via a communication interface.
  • the network can be the Internet, an Internet and/or an extranet, or an intranet and/or extranet in communication with the Internet.
  • the network is a telecommunications and/or data network.
  • the network can include one or more computer servers that can implement distributed computing, such as cloud computing.
  • the CPU can execute a series of machine-readable instructions, which can be embodied in a program or software.
  • the instructions can be stored in a storage location, such as a memory.
  • the instructions can be directed to the CPU, which can then program or otherwise configure the CPU to implement the methods of the present disclosure. Examples of operations performed by the CPU can include fetching, decoding, executing, and writing back.
  • the CPU can be part of a circuit, such as an integrated circuit.
  • the one or more processors 102 and/or other data processing circuits described above may generally be referred to herein as "data processing circuitry.”
  • the data processing circuitry may be embodied in whole or in part as software, hardware, firmware, or any other combination thereof.
  • the data processing circuitry may be a single, independent processing module, or may be incorporated in whole or in part into any of the other components of the computer terminal 10 (or mobile device).
  • the data processing circuitry functions as a processor control (e.g., selection of a variable resistor terminal path connected to an interface).
  • the memory 104 can be used to store software programs and modules of application software, such as the program instructions/data storage device corresponding to the method for detecting samples in the embodiment of the present disclosure.
  • the processor 102 executes various functional applications and data processing by running the software programs and modules stored in the memory 104, that is, implementing the vulnerability detection method of the above-mentioned application.
  • the memory 104 may include a high-speed random access memory, and may also include a non-volatile memory, such as one or more magnetic storage devices, flash memory, or other non-volatile solid-state memory.
  • the memory 104 may further include a memory remotely located relative to the processor 102, and these remote memories may be connected to the computer terminal 10 via a network. Examples of the above-mentioned network include, but are not limited to, the Internet, an intranet, a local area network, a mobile communication network, and combinations thereof.
  • the transmission device 106 is used to receive or send data via a network.
  • Specific examples of the aforementioned network may include a wireless network provided by the communication provider of the computer terminal 10.
  • the transmission device 106 includes a network interface controller (NIC), which can be connected to other network devices via a base station to communicate with the Internet.
  • the transmission device 106 may be a radio frequency (RF) module, which is used to communicate with the Internet wirelessly.
  • NIC network interface controller
  • RF radio frequency
  • the display may be, for example, a touch screen liquid crystal display (LCD) that enables a user to interact with a user interface of the computer terminal 10 (or mobile device), but is not limited thereto.
  • LCD liquid crystal display
  • control system includes at least one interaction unit configured to obtain a first instruction from a user to select at least one of at least two omics analyses.
  • 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 the detection results obtained by different detection modes.
  • sequencing data can be used for genomic analysis or transcriptomics analysis
  • tissue sample staining data can be used for pathological omics, proteomics or spatial omics analysis, etc.
  • the interaction unit can be a physical computer, keyboard, mouse, touch screen, etc., or a virtual machine, with the purpose of enabling information exchange between the machine and the user, and the specific form is not limited.
  • the unit for interacting with the multi-mode detector and the user is a first interaction unit, and the unit for interacting with the workstation and the user is a second interaction unit.
  • the multi-mode detector and the workstation can communicate via a local area network transmission method, for example, by directly connecting to a network hard disk via a network cable using the TCP/IP protocol, but is not limited thereto.
  • omics analysis refers to the analysis of at least one of the genome, cell group, proteome, metabolome, transcriptome, and epigenetic group. Taking genome and proteome analysis as an example, the inventive concept of one embodiment of the present disclosure is introduced.
  • the first instruction can be an instruction for the user to click on the controls of different software or functions, such as files in base recognition and image conversion information analysis formats, such as Cal files, BCL files or FastQ files; it can also be an instruction to click on the controls of bright field or dark field staining software or functions.
  • different software or functions such as files in base recognition and image conversion information analysis formats, such as Cal files, BCL files or FastQ files; it can also be an instruction to click on the controls of bright field or dark field staining software or functions.
  • the carrier can be a platform for realizing functions such as temperature control, chip or chip and chip frame placement, chip fluid injection or suction, and data collection in cooperation with the optical unit.
  • the fluid system further includes a reaction unit for performing a biochemical reaction on the sample.
  • the reaction unit can realize biochemical reactions for sequencing, as well as biochemical processes such as immunohistochemistry and multiple staining imaging.
  • the device further includes at least one processor coupled to the interaction unit, the carrier, and the reaction unit, and configured to:
  • the user is instructed to input initial information, where the user input method 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, reagent kit number and confirmation information.
  • the user input method 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
  • the initial information includes at least one of chip type, sequencing scheme, staining scheme, reagent kit number and confirmation information.
  • the code scanning input can be completed by inputting the identification code on the carrier or chip using an external code scanning gun, or by inputting the identification code on the carrier through an optical unit, and there is no restriction here.
  • the sequencing scheme may be any one of Sanger sequencing, NGS sequencing, single molecule sequencing, and nanopore sequencing, but is not limited thereto.
  • the staining scheme refers to a staining scheme including the antibody name, wavelength and staining time or a nucleic acid probe fluorescence detection scheme.
  • the kit number refers to the different codes for different types of kits.
  • confirmation information refers to the confirmation of the information entered by the user. For example, when performing protein analysis, it confirms the input antibodies, time and other information to avoid users finding input errors and clicking to directly enter the next step, which can effectively prevent mistakes.
  • the processor is further configured to control the reaction unit to perform a biochemical reaction according to the initial information.
  • the processor is further configured to control the interaction unit to feed back the information being processed by the user.
  • users can access images in stages. For example, after a preset number of cycles have been sequenced, a sequencing image or result may be displayed, or a "Sequencing in Progress" interface may be displayed without displaying the sequencing image or result.
  • a sequencing image or result may be displayed, or a "Sequencing in Progress" interface may be displayed without displaying the sequencing image or result.
  • the interactive unit displaying images after each round of brightfield or darkfield staining, or displaying the countdown to the completion of a biochemical reaction. This allows the interactive unit to provide feedback to the user regarding the information being processed.
  • each round of scanning will display an image after the current round of staining. If the user determines that the staining result or staining quality does not meet expectations, the biochemical reaction process can be interrupted or terminated.
  • the processor is further configured to complete the biochemical reaction based on the reaction unit and generate a first processing result.
  • the first processing result can be the Cal file, BCL file, FastQ file, offline report, quality control report and other contents after sequencing is completed, but is not limited to this. It can also be a highly multiplexed map of the cell types and pathological characteristics of animals or plants after multiple cycles of staining, imaging and antibody elution. It can also be the result of processing the transcriptome, metabolome, and epigenetic group, such as expression statistics, differential expression analysis, functional enrichment analysis, metabolite identification, metabolic pathway analysis, methylation level analysis, ChIP-seq analysis, function and pathway analysis and other data, information or reports.
  • the processor is further configured to control the interaction unit to feed back the first processing result to the user.
  • the first processing result is data, information or a report, which can be fed back to the user through display screen, voice prompts and brain-computer interface output, but is not limited to these.
  • integrated circuits such as CMOS FETs and biosensors
  • PCI peripheral component interconnect
  • FPGAs field programmable gate arrays
  • ASICs application-specific integrated circuits
  • 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 that 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 handle large-scale computing tasks that can be processed in parallel.
  • cloud platforms can provide the necessary support.
  • local FPGA/ASIC chipsets and CPUs/GPUs can provide the required computing power.
  • the at least one interaction unit further includes obtaining a second instruction input by the user.
  • the first instruction and the second instruction input by the user can be obtained through only one interaction unit, or the first instruction and the second instruction input by the user can be obtained respectively through different interaction units. It can be set according to actual needs and depends on the hardware design, which will not be repeated here.
  • the second instruction may be an instruction for the user to click on primary analysis, secondary analysis, or tertiary analysis, or may be a control for clicking on software or functions of cell analysis or protein analysis, but is not limited thereto.
  • the processor is further configured to instruct the user to input analysis information according to the second instruction, where the analysis information includes at least one of opening a file to be analyzed, selecting an area of interest, and selecting an analysis target.
  • the file to be analyzed can be a report from the machine after the sequencing is completed, a sequencing file, or a protein or cell image set that has undergone multiple rounds of staining. Opening the file to be analyzed can include the process of loading colors into the protein or cell image set. Selecting the region of interest can determine the scope of cell segmentation and analysis. It can be selected automatically or manually according to user needs. Selecting the analysis target can be selecting different markers to determine the number and probability of cells corresponding to the marker when the table is finally generated. It can also be a control displayed on the interface corresponding to the primary analysis of the sequencing data, the conversion of Cal files or BCL files to FastQ files, primary analysis, secondary analysis, tertiary analysis, and diagnostic analysis to determine what kind of analysis to perform subsequently.
  • the processor is also used to analyze the first processing result and generate a second processing result according to a third preset method.
  • the third preset method includes converting the Cal file or BCL file into a FastQ file, primary analysis, secondary analysis, tertiary analysis, diagnostic analysis, merging all images in the first processing result, displaying in the interactive unit, performing cell segmentation according to the region of interest, analyzing the segmented cells, and generating at least one of information including the number of cells corresponding to the marker and the positive probability.
  • the processor is further used to control the interactive unit to feed back the second processing result to the user, where the second processing result includes at least one of a secondary analysis result, a tertiary analysis result, a quantitative analysis, a phenotypic analysis, a spatial analysis, and a diagnostic report.

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Abstract

La présente divulgation concerne un système de détermination ayant de multiples modes de détection et un procédé associé. Le système de détermination ayant de multiples modes de détection comprend : un étage d'échantillon ; un module optique utilisé pour balayer un échantillon à l'essai, le module optique ayant au moins un mode de séquençage et un mode de coloration par fluorescence ; un circuit d'attaque couplé sélectivement à des paramètres de mode correspondant à un mode de fonctionnement du module optique, les paramètres de mode comprenant au moins un premier paramètre de commande de mode correspondant au mode de séquençage et un second paramètre de commande de mode correspondant au mode de coloration par fluorescence ; et un circuit de commutation de mode couplé au circuit d'attaque pour commander le circuit d'attaque sur la base de l'échantillon testé : le circuit de commutation de mode associe différents paramètres de mode au circuit d'attaque sur la base du type de l'échantillon testé. La présente divulgation permet d'obtenir une commutation multimode du système de détection, et permet d'obtenir de multiples fonctions de détection sur une même plateforme.
PCT/CN2025/089147 2024-04-19 2025-04-15 Système de détermination ayant de multiples modes de détection, procédé de détection et procédé de commutation de mode associé Pending WO2025218675A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06289017A (ja) * 1993-04-06 1994-10-18 Matsushita Electric Ind Co Ltd Ｄｎａの塩基配列決定方法及びｄｎａの塩基配列決定用測定装置
US20040029213A1 (en) * 2000-06-08 2004-02-12 Callahan Daniel E. Visual-servoing optical microscopy
JP2008209383A (ja) * 2007-02-01 2008-09-11 Sysmex Corp 検体分析装置
US20140073517A1 (en) * 2010-02-23 2014-03-13 Rheonix, Inc. Self-contained biological assay apparatus, methods, and applications
CN110032061A (zh) * 2018-01-12 2019-07-19 伊鲁米那股份有限公司 实时控制器切换
CN113834803A (zh) * 2021-09-22 2021-12-24 福州大学 一种多功能显微成像光学系统

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06289017A (ja) * 1993-04-06 1994-10-18 Matsushita Electric Ind Co Ltd Ｄｎａの塩基配列決定方法及びｄｎａの塩基配列決定用測定装置
US20040029213A1 (en) * 2000-06-08 2004-02-12 Callahan Daniel E. Visual-servoing optical microscopy
JP2008209383A (ja) * 2007-02-01 2008-09-11 Sysmex Corp 検体分析装置
US20140073517A1 (en) * 2010-02-23 2014-03-13 Rheonix, Inc. Self-contained biological assay apparatus, methods, and applications
CN110032061A (zh) * 2018-01-12 2019-07-19 伊鲁米那股份有限公司 实时控制器切换
CN113834803A (zh) * 2021-09-22 2021-12-24 福州大学 一种多功能显微成像光学系统

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