WO2021120288A1 - Puce d'analyse micrototale, ensemble puce et procédé de préparation d'échantillon de cellule unique - Google Patents

Puce d'analyse micrototale, ensemble puce et procédé de préparation d'échantillon de cellule unique Download PDF

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Publication number
WO2021120288A1
WO2021120288A1 PCT/CN2019/129020 CN2019129020W WO2021120288A1 WO 2021120288 A1 WO2021120288 A1 WO 2021120288A1 CN 2019129020 W CN2019129020 W CN 2019129020W WO 2021120288 A1 WO2021120288 A1 WO 2021120288A1
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cell
port
label
chip
microchannel
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PCT/CN2019/129020
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English (en)
Chinese (zh)
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戴敬
丁志文
张惠丹
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苏州昊通仪器科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biological instruments and equipment, and more specifically to a micro-total analysis chip, a chip assembly and a single-cell sample preparation method for preparing single-cell samples.
  • Single-cell transcriptomics is an effective method for elucidating complex biological systems. This method studies the gene expression of individual cells through a microscopic perspective.
  • Single-cell RNA sequencing can analyze specific differences between different types of cells in complex samples by generating single-cell maps of cell populations.
  • single-cell gene expression profiling also brings important insights into cell processes in the development of diseases. Advances in single-cell sequencing technology allow researchers to obtain key data that was often obscured by bulk cell sequencing methods, such as rare or new cell types, so as to explore the true diversity of gene expression at the single-cell level .
  • the preparation of qualified single-cell samples is the key to the successful application of the technology.
  • the Phapsody single cell analysis system of BD Company of the United States uses flat array microwells for cell capture and performs 3'RNAseq determination based on microspheres.
  • a cell is matched with a barcode-labeled microsphere, then the cell is lysed, the mRNA is hybridized with the barcode-labeled capture oligonucleotide on the microsphere, the microsphere is recovered by magnetic force and cNDA synthesis is performed, followed by sequencing and construction Single cell gene expression profile.
  • This method requires stricter cell size and morphology. Cells that are too large, such as egg cells, nerve cells, and cardiomyocytes, are difficult to load into the micropores, resulting in extremely low effective cell trapping flux; Cross-contamination, thereby increasing the system noise and accuracy of single-cell analysis.
  • the C1 single cell analysis system of Fluidigm, USA uses a microvalve array structure to trap cells in an isolated cavity. This method also has the problem of cross-contamination between the cells to be analyzed. In addition, the throughput of this method is low, and only about 800 cells can be captured and analyzed each time.
  • micro-total analysis chips also called microfluidics.
  • Chinese Patent CN104350374B discloses a method, system and device for capturing and processing multiple single cells using microfluidics.
  • the use of the micro-total analysis chip can isolate cells and cells by a specific fluid medium to avoid cross-contamination between cells; and the continuous movement of cells in the micro-channels improves the preparation throughput.
  • the manufacturing cost of the micro whole analysis chip is relatively high, and in the actual use process, there is also a phenomenon that the quality of the obtained cells is not high.
  • the purpose of the present invention is to overcome or at least alleviate the above-mentioned shortcomings of the prior art, and to provide a micro total analysis chip, a chip assembly and a single-cell sample preparation method that can obtain a higher-quality single-cell sample.
  • a micro-total analysis chip for preparing single-cell samples which includes at least two inflow ports, one outflow port, and a plurality of outflow ports connecting the inflow port and the outflow port. Tao, where,
  • the inflow port is used for allowing the cell suspension, molecular label and cell isolation medium to enter the multiple flow channels, and the outflow port is used for allowing the single cell sample to flow out of the flow channel,
  • the inflow port is located on the upper surface of the chip, and the outflow port is located on the lower surface of the chip.
  • the single cell sample can flow out of the outflow port by gravity.
  • the inflow port includes a cell and label port and an isolation medium port, the cell and label port is used to load cells and molecular label carrier fluid, and the isolation medium port is used to load a cell isolation medium,
  • the multiple flow channels include label cell micro flow channels, isolation medium micro flow channels and single cell micro flow channels,
  • the label cell microchannel is connected with the cell and the label port
  • the isolation medium microchannel is connected with the isolation medium port
  • the label cell microchannel and the isolation medium microchannel are converged at a junction
  • the single-cell microfluidic channel connects the confluence port and the outflow port.
  • the label cell microchannel includes a first label cell microchannel, a second label cell microchannel, and a third label cell microchannel
  • the first label cell microchannel and the second label cell microchannel are connected by a buffer zone, and a plurality of obstacles are arranged in the buffer zone to disperse the cells when passing through the buffer zone,
  • a third cell microchannel is connected between the first junction and the isolation medium microchannel.
  • the inflow port includes a cell port, a label port, and an isolation medium port
  • the cell port is used for loading cell suspension
  • the label port is used for loading molecular tags
  • the isolation medium port is used for Load cell isolation medium
  • the multiple flow channels include cell micro flow channels, label micro flow channels, isolation medium micro flow channels, label cell micro flow channels and single cell micro flow channels,
  • the cell microchannel is connected with the cell port
  • the label microchannel is connected with the label port
  • the isolation medium microchannel is connected with the isolation medium port
  • the cell microchannel and the label microchannel merge at a first confluence port, the first confluence port is connected to the label cell microchannel, and the label cell microchannel is downstream of the isolation medium microchannel.
  • the flow channel converges at a second junction, and the single-cell micro flow channel connects the second junction and the outflow outlet.
  • the first junction is located between the two cell microchannels, and/or
  • isolation medium micro-channels There are two isolation medium micro-channels, and the second junction is located between the two isolation medium micro-channels.
  • a chip assembly characterized in that it includes the micro total analysis chip according to the present invention, and further includes a substrate,
  • the inside of the substrate has an upstream substrate microchannel and a downstream substrate microchannel that are matched with the chip, the chip can be mounted on the substrate, and the upstream substrate microchannel is connected to the chip of the chip.
  • the inflow port and the downstream substrate micro-channel are connected to the outflow port of the chip,
  • Each of the upstream substrate microchannels has at least two openings open on the upper surface of the substrate, and each of the downstream substrate microchannels has a sample outlet opening on the lower surface of the substrate,
  • the opening is used to load cell suspension, molecular tags and cell isolation media, and the sample outlet is used to release the single cell sample.
  • N chips there are N chips, and N is greater than or equal to 2,
  • N chip mounting grooves for mounting the chip are formed inwardly on the upper surface of the substrate, and each of the chip mounting grooves is used for mounting one chip,
  • Each chip corresponds to an upstream substrate micro-channel and a downstream substrate micro-channel.
  • the chip assembly further includes a memory, which is an electronic memory capable of writing and reading information, and the memory is used to record usage information of each chip.
  • the chip assembly further includes a sample addition cup, one of the sample addition cups is provided above each of the openings, and the sample addition cup is provided at a position communicating with the upstream substrate microchannel.
  • a diversion port There is a diversion port, and the diversion port is used to allow cell suspension or molecular label or cell isolation medium to flow into the upstream substrate micro-channel under the control of the gas path module.
  • the chip assembly further includes a collection container connected to the substrate through a bracket, and the collection container is used to collect the single cell sample flowing out of the sample outlet.
  • a single-cell sample preparation method characterized in that the single-cell sample is prepared using the chip assembly according to the present invention, and the method includes:
  • the molecular tag is mixed with the cell suspension to form a cell and molecular tag carrier fluid
  • the cell isolation medium acts on the cell and molecular label carrier fluid, and the carrier fluid having a continuous liquid phase becomes a dispersed liquid phase under the action of the shear force from the cell isolation medium, and the single cell Be dispersed and isolated;
  • a method for preparing a single-cell sample characterized in that the single-cell sample is prepared by using the chip assembly according to the present invention, and the method includes:
  • the information corresponding to each chip is read from the memory, and after each chip is used, the information of the chip stored in the memory is updated.
  • the micro-total analysis chip according to the present invention has a simple structure and can prepare high-quality single-cell samples.
  • the manufacturing cost is low, and the single cell preparation efficiency and the quality of the obtained single cells can be improved.
  • the single-cell sample preparation efficiency is high, the obtained single-cell sample is of high quality, and no contamination occurs between samples.
  • Fig. 1 is an exploded schematic diagram of a part of the structure of a chip assembly according to an embodiment of the present invention.
  • Fig. 2 is a top view of a chip assembly according to an embodiment of the present invention.
  • Fig. 3 is a schematic view taken along the axial direction of a part of the structure of a chip assembly according to an embodiment of the present invention.
  • FIG. 4 is an exploded schematic diagram of a partial structure of a single-cell sample preparation and processing device according to another embodiment of the present invention.
  • Fig. 5 is a schematic diagram of a microfluidic channel of a chip according to an embodiment of the present invention.
  • Fig. 6 is a schematic diagram of a micro flow channel of a chip according to another embodiment of the present invention.
  • Fig. 7 is a schematic flow chart of a method for preparing a single cell sample according to an embodiment of the present invention.
  • Fig. 8 is a schematic diagram of a partial structure of a single-cell sample preparation and processing device according to an embodiment of the present invention.
  • Fig. 9 is a schematic block diagram of each part of a single-cell sample preparation and processing device according to an embodiment of the present invention.
  • Fig. 10 is a schematic diagram of an optical path module of a single-cell sample preparation and processing device according to an embodiment of the present invention.
  • a cell flow direction a cell flow direction; b label flow direction; c isolation medium flow direction; d single cell sample flow direction; e labeled cell flow direction;
  • FIG. 1, FIG. 3, and FIG. 4 is used to define the position of "up” and “down” in the chip and chip assembly according to the present invention.
  • the chip assembly according to the present invention includes a substrate 10, a plurality of (eight in FIG. 1) chips 20 (ie, micro-full analysis chips), and a memory 60.
  • the upper surface of the substrate 10 is provided with a recessed chip mounting groove 101 for mounting the chip 20. It should be understood that the position of the chip mounting groove 101 is only schematically shown in FIG. 1, and the position of the chip mounting groove 101 is not shown in proportion. Shape and size. Each chip mounting slot 101 is used to mount a chip 20.
  • the inside of the substrate 10 has multiple groups of substrate micro-channels, and the inside of the chip 20 has chip micro-channels (the specific structure of the chip micro-channels will be further described with reference to FIGS. 5 and 6). Each group of substrate micro-channels is connected to one The chip micro-channels of the chip 20 are connected, so that cells, molecular tags (also referred to as labels) and cell isolation media (also referred to as isolation media for short) can circulate between the substrate micro-channels and the chip micro-channels.
  • Each group of substrate microchannels includes an upstream substrate microchannel and a downstream substrate microchannel, and the chip 20 is connected between the upstream substrate microchannel and the downstream substrate microchannel.
  • Each upstream substrate microfluidic channel has two or three inlets opened on the upper surface of the substrate 10.
  • FIG. 4 shows a solution with two inlets on the upper surface of the substrate 10, one of which is used for the isolation medium to enter the substrate microfluidic channel, and the other is used for the cell and molecular label carrier fluid to enter the substrate microfluidic. Tao.
  • FIG. 1 shows a scheme in which the upper surface of the substrate 10 has three inlets, and the three inlets are respectively used for the isolation medium, the cell suspension and the molecular tag to enter the substrate microfluidic channel.
  • each inlet for the cell suspension is in communication with a cell sampling cup 31
  • each inlet for the isolation medium is in communication with an isolation medium sampling cup 41
  • each inlet for the molecular tag enters. It communicates with a label adding cup 51.
  • a larger dose of cells, isolation media, and molecular tags can be added to the chip for processing at one time.
  • the way of adding samples can be manual sample addition by the operator or automatic sample addition by the instrument.
  • the sample addition cup is provided with a diversion port at the connection with the upstream substrate micro flow channel, so that the cell suspension or molecular label or isolation medium can flow into the micro flow channel under the control of the gas path module (not shown).
  • molecular tags and isolation media flow through the microfluidic channel of the chip, a molecular tag and a cell are combined to form a labeled cell.
  • the solution at this time is called the cell and molecular tag carrier fluid; then there is a continuous liquid phase
  • the cell and molecular label carrier fluid is sheared by the isolation medium into a cell and molecular label carrier fluid in a dispersed liquid phase, that is, single cells are dispersed and isolated to obtain a single cell sample.
  • a cell sample addition cup cover 32 is provided for each cell sample addition cup 31, an isolation medium sample addition cup cover 42 is provided for each isolation medium sample cup 41, and a label sample addition cup is provided for each label sample cup 51 Cover 52.
  • each inlet for the isolation medium to enter is communicated with an isolation medium sampling cup 41, and each inlet for the cell and molecular label carrier fluid to enter. It communicates with a cell and label adding cup 531.
  • the cell and label adding cup 531 contains both the cell suspension and the molecular label.
  • a cell and label sample cup cover 532 is provided for each cell and label sample cup 531.
  • Each downstream substrate microfluidic channel has a sample outlet 103 opening downward on the lower surface of the substrate 10, and the single-cell sample that has been marked and isolated can flow out of the sample outlet 103 under the action of gravity.
  • Single-cell samples obtained by gravity are less prone to damage than single-cell samples obtained with other tools, and the obtained single-cell samples are of higher quality.
  • the chip assembly further includes a collection container 70 having a test tube shape (see FIG. 4 ), and the collection container 70 is connected to the substrate 10 through a bracket so that the single cell sample flowing out of the sample outlet 103 can flow into the collection container 70.
  • the collection container 70 is a transparent container to facilitate the operation of separating the molecular tag from the carrier through the action of the light field, which will be described below.
  • the lower surface of the substrate 10 is provided with a substrate lower sealing sheet 104 and a lower sealing gasket 105 in sequence.
  • multiple chips 20 contained in a chip assembly may be different, that is, these chips 20 may have different chip microfluidics, so that one chip assembly can be used to process multiple different samples; The same chip 20 can also be used to process different samples as needed. Therefore, the way of integrating multiple chips 20 on one substrate 10 increases the processing capacity of the chip assembly for samples.
  • the substrate micro-channels provided on the substrate 10 may be the same.
  • each chip component can process multiple different samples.
  • each chip component contains multiple chips 20.
  • the information is recorded in the memory 60 in advance.
  • the substrate 10 has a memory mounting groove 102 to mount the memory 60.
  • the memory 60 is an electronic memory with readable and writable functions.
  • the memory 60 may be an electronically erasable memory (EEPROM), a flash memory (FLASH), a ferroelectric memory (FRAM), or a solid state drive (SSD).
  • EEPROM electronically erasable memory
  • FLASH flash memory
  • FRAM ferroelectric memory
  • SSD solid state drive
  • each chip 20 included in the chip assembly can be obtained by reading the information in the memory 60 with the help of an electronic device.
  • new information can be written into the memory 60 with the help of electronic equipment, so as to facilitate the recording of the usage of each chip 20.
  • one chip assembly can be used for multiple sample processing experiments, and each sample processing experiment uses a part of the unused chips 20 in the chip assembly, so as to ensure that each chip 20 is not reused.
  • FIG. 5 shows the structure of the micro flow channel corresponding to the chip 20 having three inlets for the upstream substrate micro flow channel.
  • the micro flow channel of the chip 20 includes three inlets corresponding to the three inlets of the upstream substrate micro flow channel, namely, the cell port 11, the label port 12, and the isolation medium port 13.
  • the chip micro flow channel It also includes an outflow port 14 for connecting to the downstream substrate micro-channel.
  • the inflow port is located on the upper surface of the chip 20, and the outflow port 14 is located on the lower surface of the chip 20.
  • Each branch flow channel connects the above-mentioned three inflow ports and one outflow port together.
  • two branched symmetrically arranged cell microchannels f1 are separated.
  • Each cell microchannel f1 is arranged tortuously, so that the flow resistance of the flow channel has a change in the flow path of the cell microchannel f1, and the liquid flow rate of the cell suspension will change with the flow resistance.
  • the flow resistance changes on the flow path enable unevenly dispersed cells to be gradually dispersed uniformly under the adjustment of the liquid carrier flow, avoiding cell aggregation or agglomeration.
  • the arrow a in Fig. 5 shows the cell flow direction.
  • a label microchannel f2 is formed.
  • the length of the label microchannel f2 is smaller than the length of the cell microchannel f1.
  • the label microchannel f2 is located between the two cell microchannels f1, and it merges with the two cell microchannels f1 at the first confluence port C1.
  • the angle between the label microchannel f2 and the two cell microchannels f1 is both 90°.
  • isolation medium port 13 two branched symmetrically arranged isolation medium microchannels f3 are separated.
  • the isolation medium microchannel f3 extends toward the first confluence port C1 and merges with the label cell microchannel f4 at a second confluence port C2 downstream of the first confluence port C1.
  • the label cell microchannel f4 is located between the two isolation medium microchannels f3, and at the second junction C2, the angle between the label cell microchannel f4 and the two isolation medium microchannels f3 is 90. °.
  • the isolation medium separates the labeled cells one by one in a vertical cutting manner, so that the carrier fluid having a continuous liquid phase becomes a carrier fluid of a dispersed liquid phase, and a single cell sample is obtained.
  • the arrow c in FIG. 5 shows the flow direction of the isolation medium.
  • the inner diameters of the micro flow channels at the first junction C1 and the second junction C2 are smaller than the inner diameters of the micro flow channels at other paths, also called micro flow at the first junction C1 and the second junction C2
  • the tract forms a constriction. The necking makes the micro flow channels converge so that the cells in the flow channels can be labelled or isolated one by one.
  • the inner diameter of the single-cell microchannel f5 is larger than the inner diameter of the microchannels in other parts of the chip 20.
  • the cell port 11, the cell microchannel f1, the label port 12, and the label microchannel f2 are located on one side, and the isolation medium port 13 and the isolation medium microchannel f3 are located on the other side, This is not necessary.
  • the isolation medium port 13 and the isolation medium microchannel f3 may also be located on the same side as the cell port 11.
  • the number of cell microchannels f1 and isolation medium microchannels f3 may not be limited to two, for example, there may be only one cell microchannel f1.
  • the present invention is suitable for the cell port 11, the label port 12, the isolation medium port 13, the outflow port 14, the cell microchannel f1, the label microchannel f2, the isolation medium microchannel f3, the label cell microchannel f4, and the single cell microflow channel.
  • the specific arrangement position and shape of the road f5 are not limited.
  • FIG. 6 shows the structure of an embodiment of the micro flow channel corresponding to the chip 20 having two inlets for the upstream substrate micro flow channel.
  • the micro flow channel of the chip 20 includes two inlets respectively corresponding to the two inlets of the upstream substrate micro flow channel, namely the cell and label port 111 and the isolation medium port 13, and the chip micro flow channel also includes An outflow port 14 for connecting to the micro flow channel of the downstream substrate.
  • the inflow port is located on the upper surface of the chip 20, and the outflow port 14 is located on the lower surface of the chip 20.
  • the cell and the molecular tag are mixed together to complete the combination of the molecular tag and the cell in advance, that is, the cell and the molecular tag carrier fluid are already entering the cell and tag port 111.
  • the cell and label port 111 is connected to the first label cell microchannel f11 to transport the labeled cells downstream.
  • the downstream end of the first label cell microchannel f11 is connected to the buffer C0.
  • the labeled cells can be scattered and evenly distributed by obstacles.
  • second label cell microchannels f12 are connected downstream of the buffer C0.
  • the inner diameter of the second label cell microchannel f12 is smaller than the inner diameter of the first label cell microchannel f11, and When the labeled cells flow through the second labeled cell microchannel f12, they pass through the cross section of the microchannel singly.
  • the arrow e in Fig. 6 shows the flow direction of the cell and molecular tag carrier fluid.
  • a plurality of second label cell microchannels f12 first converge at the first confluence port C1, and a third label cell microchannel f13 is formed downstream of the first confluence port C1, and the third label cell microchannel f13 will be isolated and isolated downstream Intersection of media. It should be understood that the length of the third label cell microchannel f13 can be very short.
  • isolation medium port 13 two branched symmetrically arranged isolation medium microchannels f3 are separated.
  • the isolation medium microchannel f3 extends toward the first confluence port C1, and merges with the third label cell microchannel f13 at the second confluence port C2 downstream of the first confluence port C1.
  • the third label cell microchannel f13 is located between the two isolation medium microchannels f3.
  • the isolation medium is cut in from the side of the cell flow direction to isolate the labeled cells one by one to form a single-cell sample.
  • the arrow c in FIG. 6 shows the flow direction of the isolation medium.
  • a simple summary of the method for preparing a single-cell sample using the micro-total analysis chip or chip assembly according to the present invention includes:
  • the single cell sample flowing into the collection container 70 is in the form of droplets.
  • the molecular tag exists in the form of being bound to a certain carrier (for example, molecular tag-carrying microspheres or magnetic beads).
  • a certain carrier for example, molecular tag-carrying microspheres or magnetic beads.
  • NGS Next Generation Sequencing
  • the single-cell sample preparation and processing device includes an electrical control system 1, a chip assembly 2, a power supply 3, an operation display unit 4, and multiple functional modules (including a chip loading module 5.1, a sensor module 5.2, heating and Refrigeration module 5.3, electrode module 5.4, gas circuit module 5.5, liquid circuit module 5.6 and optical circuit module 5.7).
  • the electrical control system 1 is connected to a power source 3 and includes a plurality of sub-units for controlling the work of each functional module.
  • the electrical control system 1 includes a dedicated computer 1.1, a motion control unit 1.2, a sample detection unit 1.3, a temperature control unit 1.4, an inverter high voltage generating unit 1.5, a gas circuit control unit 1.6, a liquid circuit control unit 1.7, and a light field generating unit 1.8 .
  • the dedicated computer 1.1 is electrically connected to the operation display unit 4 for visually inputting or outputting instructions.
  • the motion control unit 1.2 is used to control the chip loading module 5.1.
  • the chip loading module 5.1 includes, for example, a motor, a moving link, and a support H (refer to FIG. 8 at the same time).
  • the operator places the substrate 10 loaded with the chip 20 and each sample cup on the support H of the chip loading module 5.1.
  • the support H can Under the control of the motion control unit 1.2, it is moved to a designated working position, and the substrate 10 is positioned above the collection container 70 to ensure the accurate development of subsequent processing operations.
  • the sample detection unit 1.3 is used to receive the signal from the sensor module 5.2, so as to provide control parameters for the system.
  • the sensor module 5.2 can include flow rate sensors, pressure sensors, and liquid level sensors.
  • the flow rate sensor is used to measure the flow rate of the liquid flowing through the microchannels of the chip assembly, and the pressure sensor and the liquid level sensor are respectively used to detect each container (for example, including the cell sample adding cup 31, the isolation medium sample adding cup 41, and the label sample adding cup 31).
  • the temperature control unit 1.4 is used to control the temperature provided by the heating and cooling module 5.3, so as to provide a suitable reaction temperature for the working position where the chip assembly 2 is located.
  • the gas path control unit 1.6 is used to control the amount of gas provided by the gas path module 5.5, so that the cell sample cup 31, the isolation medium sample cup 41, the label sample cup 51, or the cell and the label sample cup 531 It can flow into the micro flow channel of the chip assembly 2 more smoothly.
  • the liquid path control unit 1.7 is used to control the liquid path module 5.6.
  • the liquid path module 5.6 for example, is used to automatically add the isolation medium to the isolation medium sample adding cup 41, which simplifies the operation of the operator.
  • the light field generating unit 1.8 is used to control the light path module 5.7 to provide the collection container 70 of the chip assembly 2 with a light field of suitable energy, so that the molecular label of the single cell sample in the collection container 70 is separated from its carrier.
  • the optical path module 5.7 may be shown in Fig. 10, for example, which includes a light source 5.71, a collimator lens 5.72, a reflector 5.73, a focusing lens 5.74, an optical fiber 5.75, a diffuser 5.76, an aperture 5.77, and a telephoto system 5.78.
  • the light source 5.71 is, for example, an LED light source, a laser light source, an ultraviolet light source or a mercury lamp.
  • the wavelength peak of the light wave generated by the light source 5.71 is preferably between 254 nm and 450 nm.
  • the light emitted from the light source 5.71 sequentially passes through the collimator lens 5.72, the reflector lens 5.73, the focusing lens 5.74, the optical fiber 5.75, the diffuser sheet 5.76, the diaphragm 5.77 and the telephoto system 5.78 to illuminate the collection container 70.
  • the aperture size of the diaphragm 5.77 and the specifications of the lenses in the telephoto system and the distance between them the size of the light spot and the size of the light energy irradiated to the collection container 70 can be controlled.
  • the molecular label in the droplet will be separated from the carrier, and the separated molecular label will be free in the liquid, which increases the probability of the poly T on the label and the poly A tail of the mRNA pairing and binding.
  • the inverter high voltage generating unit 1.5 controls the electrode module 5.4 to apply an electric field to the droplets in the collection container 70.
  • a first electrode 81 and a second electrode 82 are provided on the outer periphery of the collection container 70.
  • the first electrode 81 and the second electrode 82 are spaced apart in the vertical direction so that the electric field formed between the first electrode 81 and the second electrode 82 has The component in the vertical direction.
  • the upper area of the outer circumference of each collection container 70 has an annular first electrode ring 810, and the lower area of the outer circumference of each collection container 70 has a cylindrical second electrode. ⁇ 820.
  • the height of the second electrode ring 820 is equal to or greater than the height of the portion of the collection container 70 located below the first electrode ring 810 (referred to as the lower half of the collection container 70), so that the lower half of the collection container 70 can be It is completely contained in the inner cavity of the second electrode ring 820.
  • a plurality of first electrode rings 810 are connected to form a first electrode 81, and a plurality of second electrode rings 820 are connected to form a second electrode 82.
  • the second electrode ring 820 may only surround the central area of the collection container 70 in the axial direction, instead of surrounding the entire lower portion of the collection container 70.
  • the annular second electrode ring 820 is, for example, a plate with a middle hole. shape.
  • FIG. 4 shows a preferred embodiment of the first electrode 81 and the second electrode 82.
  • the first electrode 81 and the second electrode 82 may also be configured to consist of one
  • the ring surrounds the form of a plurality of collection containers 70.
  • the first electrode 81 and the second electrode 82 are made of metal and/or conductive non-metal.
  • the first electrode 81 and the second electrode 82 can be made of one of graphite, aluminum, and copper. Or more.
  • the inverter high voltage generating unit 1.5 is connected to a DC or AC power supply, and the voltage range of the power supply is 1 to 100V.
  • the input voltage of the inverter high voltage generating unit 1.5 is 24V;
  • the voltage generated by the first electrode 81 and the second electrode 82 is -10KV to +10KV AC pulse voltage, the frequency and voltage value of the AC pulse voltage can be adjusted according to the distance between the first electrode 81 and the second electrode 82 and the distance between the droplets.
  • the droplets in the collection container 70 are balanced under the action of gravity and the buoyancy of the carrier liquid, and make random motions on the microscopic level.
  • an electric field is generated between the first electrode 81 and the second electrode 82, the droplet is vibrated by the force of the electric field, so that the surface tension that maintains the spherical shape of the droplet changes drastically.
  • the electric force of the electric field is the largest.
  • the random movement of the droplet and the movement of the electric field vibration will resonate, the surface tension of the droplet will lose balance, and the droplet will have amplitude along the direction of the gravity field
  • the maximum vibration and unbalanced surface tension cause the droplets to fuse, that is, the droplets break the emulsion.
  • the molecular tags combined with cellular mRNA are mixed together to facilitate subsequent NGS library construction.
  • the molecular tag captures the mRNA of a single cell and is heated immediately after reverse transcription to synthesize the cDNA fragment, without the need for pipetting and tube transfer operations.
  • the cell and the cell are completely isolated by the isolation medium, which avoids cross-contamination between cells.
  • micro full analysis chip adopts a modular design and manufacturing method. Different chips 20 can be mounted on the same substrate 10 to obtain chip components of different specifications, and the manufacturing cost of the chip components is low.
  • One chip assembly includes multiple chips 20, so that one chip assembly can be used to process multiple samples, which improves the efficiency of sample preparation.
  • the memory 60 manages the information of the multiple chips 20 included in one chip assembly, which can effectively and efficiently record the usage status of each chip 20, and there will be no contamination between samples.
  • the single-cell sample preparation and processing device realizes droplet demulsification by applying an electric field without adding other reagents to the reaction system.
  • the single-cell sample preparation and processing device releases the molecular tag from its carrier by applying a light field, without adding other reagents to the reaction system.
  • the single-cell sample preparation and processing device integrates an automated control module. For example, the operator does not need to manually add reagents, and the amount of reagents added can be accurately controlled, saving labor and reagent costs.
  • the cells that can be used to process the micro whole analysis chip and chip assembly according to the present invention include but are not limited to: cell line cells (such as human HEK293T, mouse NIH3T3), tissue digestion cells (such as mouse brain tissue E18neuron), and human peripheral blood Monocytes (PBMC).
  • cell line cells such as human HEK293T, mouse NIH3T3
  • tissue digestion cells such as mouse brain tissue E18neuron
  • PBMC peripheral blood Monocytes
  • the diameter of the processed cells is usually 5um to 30um, but the present invention does not limit this.

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Abstract

L'invention concerne une puce d'analyse micrototale (20), un ensemble puce (2) et un procédé de préparation d'un échantillon de cellule unique. La puce d'analyse micrototale (20) est utilisée pour préparer l'échantillon de cellule unique, et comprend au moins deux orifices d'entrée, un orifice de sortie (14) et de multiples canaux d'écoulement reliant les orifices d'entrée et l'orifice de sortie (14). Les orifices d'entrée sont utilisés pour permettre l'entrée d'une suspension cellulaire, d'une étiquette moléculaire et d'un milieu d'isolement de cellules dans les multiples canaux d'écoulement ; l'orifice de sortie (14) est utilisé pour permettre l'écoulement de l'échantillon de cellule unique hors du canal d'écoulement ; les orifices d'entrée sont situés sur la surface supérieure de la puce (20) et l'orifice de sortie (14) est situé sur la surface inférieure de la puce (20). L'échantillon de cellule unique peut s'écouler hors de l'orifice de sortie (14) en fonction de l'effet de la gravité.
PCT/CN2019/129020 2019-12-20 2019-12-27 Puce d'analyse micrototale, ensemble puce et procédé de préparation d'échantillon de cellule unique WO2021120288A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120103427A1 (en) * 2009-07-09 2012-05-03 Korea Advanced Institute Of Science And Technology Multifunctional microfluidic flow control device and multifunctional microfluidic flow control method
CN105062866A (zh) * 2015-08-18 2015-11-18 深圳睿思生命科技有限公司 用于外周血循环肿瘤细胞的一次性分离芯片模块及其使用方法
CN105400679A (zh) * 2014-09-10 2016-03-16 清华大学 一种用于细胞分离的微流控芯片装置
CN105524829A (zh) * 2015-11-25 2016-04-27 成都赫尔墨斯科技有限公司 一种制造组织工程微模块的微流控芯片
CN105950469A (zh) * 2016-06-08 2016-09-21 牛海涛 细胞筛选芯片及微流控联合芯片
CN106434302A (zh) * 2016-09-18 2017-02-22 华中科技大学 一种便携式无动力源的微流控细胞分离芯片
CN108246374A (zh) * 2018-03-28 2018-07-06 东南大学 三维螺旋结构细胞分选微流控芯片及其制作方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI310835B (en) * 2006-06-23 2009-06-11 Ind Tech Res Inst Gravity-driven fraction separator and method thereof
EP3191605B1 (fr) * 2014-09-09 2022-07-27 The Broad Institute, Inc. Procédé à base de gouttelettes et appareil pour l'analyse composite d'acide nucléique de cellules uniques
CN108889352B (zh) * 2018-09-17 2023-08-22 苏州昊通仪器科技有限公司 一种高通量样本分离仪器

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120103427A1 (en) * 2009-07-09 2012-05-03 Korea Advanced Institute Of Science And Technology Multifunctional microfluidic flow control device and multifunctional microfluidic flow control method
CN105400679A (zh) * 2014-09-10 2016-03-16 清华大学 一种用于细胞分离的微流控芯片装置
CN105062866A (zh) * 2015-08-18 2015-11-18 深圳睿思生命科技有限公司 用于外周血循环肿瘤细胞的一次性分离芯片模块及其使用方法
CN105524829A (zh) * 2015-11-25 2016-04-27 成都赫尔墨斯科技有限公司 一种制造组织工程微模块的微流控芯片
CN105950469A (zh) * 2016-06-08 2016-09-21 牛海涛 细胞筛选芯片及微流控联合芯片
CN106434302A (zh) * 2016-09-18 2017-02-22 华中科技大学 一种便携式无动力源的微流控细胞分离芯片
CN108246374A (zh) * 2018-03-28 2018-07-06 东南大学 三维螺旋结构细胞分选微流控芯片及其制作方法

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