WO2021120289A1 - 单细胞样本制备和处理装置及单细胞样本微滴的处理方法 - Google Patents

单细胞样本制备和处理装置及单细胞样本微滴的处理方法 Download PDF

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WO2021120289A1
WO2021120289A1 PCT/CN2019/129023 CN2019129023W WO2021120289A1 WO 2021120289 A1 WO2021120289 A1 WO 2021120289A1 CN 2019129023 W CN2019129023 W CN 2019129023W WO 2021120289 A1 WO2021120289 A1 WO 2021120289A1
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electrode
cell sample
cell
chip
collection container
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PCT/CN2019/129023
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English (en)
French (fr)
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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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Publication of WO2021120289A1 publication Critical patent/WO2021120289A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology

Definitions

  • the invention relates to the field of biological instruments and equipment, and more specifically to a single-cell sample preparation and processing device and a single-cell sample droplet processing method.
  • Single-cell sequencing technology performs high-resolution detection of genome and transcriptome at the single-cell level, which can reveal differences in cell populations, cell evolutionary relationships, and the role of individual cells in the overall microenvironment.
  • the principle of single-cell sequencing is to amplify a small amount of whole genomic DNA and transcriptome RNA from a single cell to obtain a high-coverage, complete library, and then perform high-throughput sequencing.
  • Sample types for single-cell sequencing include, for example, tissue samples, blood PBMC (peripheral blood mononuclear cells), and cell lines. Tissue samples usually need to be physically broken, enzymatic digestion and other steps to prepare a cell suspension. After cell viability measurement and counting, reagents such as reverse transcriptase, molecular tags and PBS are added, mixed and resuspended, and then added to the cell phase cup. At the same time, add cell isolation medium and cell lysate to the cell isolation medium cup. Place the micro whole analysis chip used to prepare single cell samples in the instrument device, and the instrument control pressure module drives the cell suspension and cell isolation medium to flow into the micro whole analysis chip. Under the action of fluid shear force, separate Single cell sample droplets.
  • tissue samples usually need to be physically broken, enzymatic digestion and other steps to prepare a cell suspension.
  • reagents such as reverse transcriptase, molecular tags and PBS are added, mixed and resuspended, and then added to the cell phase cup
  • the following three steps are usually required: (i) separating the molecular tags from the carrier; (ii) heating the single-cell sample droplets so that different molecular tags capture the mRNA of a single cell, and Synthesize cDNA fragments through reverse transcription; (iii) demulsify the droplets.
  • one of the methods in the prior art is to add a certain solvent to dissolve the carrier carrier of the molecular tag, thereby freeing the molecular tag; the other method is for the molecular tag whose carrier is magnetic beads This method uses magnetic adsorption of magnetic beads, and then uses eluent to elute the molecular tags. Both of the above methods require the introduction of reagents into the reactants that are not related to the subsequent reaction, which will have a certain impact on the subsequent reaction.
  • droplet demulsification chemical methods are usually used in the prior art.
  • 10x Genomics droplet demulsification method is to add a demulsifier to the droplets.
  • Generation Sequencing (NGS) library construction inhibits, and secondly, it increases the complexity of the reaction system.
  • 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 single-cell sample preparation and processing that can prepare and process single-cell samples with a high degree of automation and without adding too many additional reagents to the reaction system.
  • Device and method for processing single cell sample droplets are provided.
  • a single-cell sample preparation and processing device which includes a chip assembly for preparing single-cell sample droplets and an electrode module for breaking the single-cell sample droplets,
  • the chip assembly includes a chip and a collection container, and the collection container is used to collect the single cell sample droplets prepared by the chip,
  • the electrode module includes a first electrode and a second electrode, the first electrode and the second electrode are both arranged on the outer circumference of the collection container, and the first electrode and the second electrode are in a vertical direction
  • the first electrode and the second electrode are at least partially overlapped in the vertical direction, so that the electric field formed between the first electrode and the second electrode has a vertical direction ⁇ The weight.
  • the first electrode is located above the second electrode
  • the first electrode includes an annular first electrode ring, and the collection container passes through the first electrode ring,
  • the second electrode includes a cylindrical second electrode ring, and the lower part of the collection container is accommodated in the inner cavity of the second electrode ring.
  • N collection containers there are N collection containers, and N is greater than or equal to 2,
  • each collection container is provided with a first electrode ring and a second electrode ring,
  • N of the first electrode rings are connected together, and N of the second electrode rings are connected together.
  • the voltage applied to the first electrode and the second electrode is an AC pulse voltage between -10KV and +10KV.
  • the device further includes an inverter high voltage generating unit, the magnitude and frequency of the voltage applied by the first electrode and the second electrode are controlled by the inverter high voltage generating unit, and
  • the inverter high voltage generating unit (1.5) is connected to a DC or AC power source, and the voltage range of the power source is 1 to 100V.
  • the material of the second electrode includes one or more of graphite, aluminum, and copper.
  • the device further includes a light path module for providing light waves that illuminate the collection container, and the light waves are used to detach the molecular labels in the single-cell sample droplets.
  • a carrier carrying the molecular tag is provided.
  • the wavelength of the light wave is between 254 nm and 450 nm.
  • the optical path module includes a diaphragm and a telephoto system, the aperture size of the diaphragm can be adjusted, and the distance between the lenses of the telephoto system can be adjusted.
  • the device further includes a liquid path module for adding reagents to the chip assembly according to a predetermined program.
  • the device further includes a sensor module, and the sensor module includes one or more of the following sensors:
  • a flow rate sensor for measuring the flow rate of the liquid flowing through the micro channel of the chip
  • a pressure sensor for measuring the pressure in each container of the chip assembly
  • the liquid level sensor is used to measure the liquid level of the liquid in each container of the chip assembly.
  • a method for processing single-cell sample droplets characterized in that the single-cell sample droplets are prepared and processed using the device according to the present invention, and the single-cell sample droplets are Housed in the collection container, the method includes:
  • the electrode module is used to apply an electric field having an electric field component in the vertical direction to the collection container, so that the single-cell sample droplet is broken by the change of surface tension under the action of the electric field, so that the single-cell sample droplet is broken.
  • the inner molecular tag is free in the single-cell sample, so as to capture the mRNA or DNA in the cell.
  • a method for processing single-cell sample droplets characterized in that the single-cell sample droplets are prepared and processed using the device according to the present invention, and the single-cell sample droplets are Housed in the collection container, the method includes:
  • the single-cell sample droplet in the collection container is irradiated with light waves, so that the molecular label in the single-cell sample droplet is separated from the carrier of the molecular label.
  • the microdroplet demulsification can be realized without adding additional reagents, so that the single-cell sequencing process is simple and the effect is good.
  • no additional reagent may be added during the process of separating the molecular label in the single-cell sample droplet from the carrier or during the process of breaking the emulsion of the droplet.
  • 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 microchannel, and the other is used for the cell and molecular label carrier fluid to enter the substrate microfluid. 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 microchannel.
  • 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-phase 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 may 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 by the light source 5.71 sequentially passes through the collimator lens 5.72, the reflecting mirror 5.73, the focusing lens 5.74, the optical fiber 5.75, the scattering 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 perform random movements on a 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 E18 neuron), and human peripheral Blood mononuclear cells (PBMC).
  • cell line cells such as human HEK293T, mouse NIH3T3
  • tissue digestion cells such as mouse brain tissue E18 neuron
  • PBMC peripheral Blood mononuclear cells
  • the diameter of the processed cells is usually 5um to 30um, but the present invention does not limit this.

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Abstract

一种单细胞样本制备和处理装置及单细胞样本微滴的处理方法，该装置包括用于制备单细胞样本微滴的芯片组件(2)和用于使单细胞样本微滴破乳的电极模块(5.4)，芯片组件(2)包括芯片(20)和收集容器(70)，收集容器(70)用于收集单细胞样本微滴，电极模块(5.4)包括设置于收集容器(70)的外周的第一电极(81)和第二电极(82)，第一电极(81)和第二电极(82)在竖直方向上间隔开、且它们在竖直方向上至少部分地重合，使得第一电极(81)和第二电极(82)之间形成的电场具有在竖直方向上的分量。

Description

单细胞样本制备和处理装置及单细胞样本微滴的处理方法
相关申请的引用
本发明要求2019年12月20日在中国提交的，名称为“单细胞样本制备和处理装置及单细胞样本微滴的处理方法”、申请号为201911326147.9的发明专利申请的优先权，该申请的全部内容通过引用并入本文。
技术领域
本发明涉及生物仪器设备领域，更具体地涉及单细胞样本制备和处理装置及单细胞样本微滴的处理方法。
背景技术
单细胞测序技术在单细胞水平对基因组、转录组进行高分辨率检测，能够揭示细胞群体差异、细胞进化关系以及单个细胞在整个微环境中的作用。
单细胞测序的原理是对分离的单个细胞微量全基因组DNA、转录组RNA进行扩增，从而获得高覆盖率的、完整的文库，之后进行高通量测序。
单细胞测序的样本类型例如包括组织样本、血液PBMC(外周血单个核细胞)和细胞系等。组织样本通常需要通过物理打碎、酶消化等步骤制备成细胞悬液，在经过细胞活性测量及计数后，加入逆转录酶、分子标签和PBS等试剂，混合重悬后加至细胞相杯。同时在细胞隔离介质杯中加入细胞隔离介质及细胞裂解液。把用于制备单细胞样本的微全分析芯片放置于仪器装置中，仪器控制压力模块驱动细胞悬液及细胞隔离介质流至微全分析芯片中，在流体剪切力的作用下，产生分离的单细胞样本微滴。
在获得单细胞样本微滴后，通常需要做以下三步的处理：(i)使分子标签脱离载体；(ii)加热单细胞样本微滴，使不同的分子标签分别捕获单一细胞的mRNA，并经过逆转录合成cDNA片段；(iii)对微滴进行破乳。
对于(i)使分子标签脱离载体，现有技术中的一种方法是添加某种溶剂，使分子标签的承载载 体溶解，从而使分子标签游离；另一种方法针对载体为磁珠的分子标签，这种方法使用磁力吸附磁珠，再用洗脱液把分子标签洗脱。上述两种方法都需要向反应物内引入与后续反应无关的试剂，会对后续反应产生一定影响。
对于(ii)加热单细胞样本微滴，现有技术通常需要把收集的单细胞样本微滴移至某一加热仪器上进行加热孵育，涉及移液、转管的操作，工序复杂。
对于(iii)微滴破乳，现有技术通常使用化学法，如10x Genomics公司的微滴破乳法是向微滴中加入破乳剂，此类方法一是会对后续的二代测序(Next Generation Sequencing,NGS)文库的构建产生抑制，二是增加了反应体系的复杂性。
发明内容
本发明的目的在于克服或至少减轻上述现有技术存在的不足，提供一种自动化程度高、且不需要向反应体系增加过多额外试剂而能制备和处理单细胞样本的单细胞样本制备和处理装置及单细胞样本微滴的处理方法。
根据本发明的第一方面，提供一种单细胞样本制备和处理装置，其包括用于制备单细胞样本微滴的芯片组件和用于使所述单细胞样本微滴破乳的电极模块，
所述芯片组件包括芯片和收集容器，所述收集容器用于收集由所述芯片制备的所述单细胞样本微滴，
所述电极模块包括第一电极和第二电极，所述第一电极和所述第二电极均设置于所述收集容器的外周，且所述第一电极和所述第二电极在竖直方向上间隔开、所述第一电极和所述第二电极在所述竖直方向上至少部分地重合，使得所述第一电极和所述第二电极之间形成的电场具有在竖直方向上的分量。
在至少一个实施方式中，所述第一电极位于所述第二电极的上方，
所述第一电极包括环形的第一电极环，所述收集容器穿过所述第一电极环，
所述第二电极包括筒形的第二电极环，所述收集容器的下部部分被容纳于所述第二电极环的内腔。
在至少一个实施方式中，所述收集容器有N个，N大于或等于2，
所述第一电极环有N个，所述第二电极环有N个，
每个所述收集容器的外周设有一个所述第一电极环和一个所述第二电极环，
N个所述第一电极环连接在一起，N个所述第二电极环连接在一起。
在至少一个实施方式中，所述第一电极和所述第二电极上加载的电压为在-10KV至+10KV之间的交流脉冲电压。
在至少一个实施方式中，所述装置还包括逆变高压发生单元，所述第一电极和所述第二电极所加载的电压的大小和频率受所述逆变高压发生单元的控制，所述逆变高压发生单元(1.5)连接直流或交流电源，所述电源的电压范围为1至100V。
在至少一个实施方式中，所述第二电极的制作材料包括石墨、铝和铜中的一者或多者。
在至少一个实施方式中，所述装置还包括光路模块，所述光路模块用于提供照射所述收集容器的光波，所述光波用于使所述单细胞样本微滴内的分子标签脱离用于承载所述分子标签的载体。
在至少一个实施方式中，所述光波的波长在254nm至450nm之间。
在至少一个实施方式中，所述光路模块包括光阑和望远系统，所述光阑的孔径大小能够被调节，所述望远系统的各透镜之间的距离能够被调节。
在至少一个实施方式中，所述装置还包括液路模块，所述液路模块用于根据预定程序向所述芯片组件添加试剂。
在至少一个实施方式中，所述装置还包括传感器模块，所述传感器模块包括以下各传感器中的一者或多者：
流速传感器，用于测量流过所述芯片的微流道的液体的流速；
压力传感器，用于测量所述芯片组件的各容器内的压力；
液位传感器，用于测量所述芯片组件的各容器内的液体的液位。
根据本发明的第二方面，提供一种单细胞样本微滴的处理方法，其特征在于，使用根据本发明的装置对所述单细胞样本微滴进行制备和处理，所述单细胞样本微滴收容于所述收集容器内，所述方法包括：
利用所述电极模块向所述收集容器施加具有在竖直方向上的电场分量的电场，使所述单细胞样本 微滴受电场作用发生表面张力的变化而破碎，从而所述单细胞样本微滴内的分子标签游离于单细胞样本中，以便于捕获细胞中的mRNA或DNA。
根据本发明的第三方面，提供一种单细胞样本微滴的处理方法，其特征在于，使用根据本发明的装置对所述单细胞样本微滴进行制备和处理，所述单细胞样本微滴收容于所述收集容器内，所述方法包括：
向所述收集容器内的所述单细胞样本微滴照射光波，使所述单细胞样本微滴内的分子标签脱离所述分子标签的载体。
根据本发明的单细胞样本制备和处理装置，能在不添加额外试剂的情况下实现微滴破乳，使得单细胞测序过程简单、效果好。
根据本发明的单细胞样本微滴的处理方法，在使单细胞样本微滴内的分子标签脱离载体的过程中、或在微滴破乳的过程中，可以不添加额外试剂。
附图说明
图1是根据本发明的一个实施方式的芯片组件的部分结构的分解示意图。
图2是根据本发明的一个实施方式的芯片组件的俯视图。
图3是根据本发明的一个实施方式的芯片组件的部分结构的沿轴向剖开的示意图。
图4是根据本发明的另一个实施方式的单细胞样本制备和处理装置的部分结构的分解示意图。
图5是根据本发明的一个实施方式的芯片的微流道的示意图。
图6是根据本发明的另一个实施方式的芯片的微流道的示意图。
图7是根据本发明的一个实施方式的单细胞样本制备方法的流程示意图。
图8是根据本发明的一个实施方式的单细胞样本制备与处理装置的部分结构的示意图。
图9是根据本发明的一个实施方式的单细胞样本制备和处理装置的各部分的示意性框图。
图10是根据本发明的一个实施方式的单细胞样本制备和处理装置的光路模块的示意图。
附图标记说明：
10基板；101芯片安装槽；102存储器安装槽；103出样口；104基板下封片；105下密封垫；
11细胞口；12标签口；13隔离介质口；14流出口；111细胞与标签口；
f11第一标签细胞微流道；f12第二标签细胞微流道；f13第三标签细胞微流道；f1细胞微流道；f2标签微流道；f3隔离介质微流道；f4标签细胞微流道；f5单细胞微流道；
C1第一汇合口；C2第二汇合口；C0缓冲区；
20芯片；31细胞加样杯；32细胞加样杯盖；41隔离介质加样杯；42隔离介质加样杯盖；51标签加样杯；52标签加样杯盖；
60存储器；70收集容器；
81第一电极；82第二电极；
a细胞流向；b标签流向；c隔离介质流向；d单细胞样本流向；e带标签的细胞流向；
5.71光源；5.72准直镜；5.73反射镜；5.74聚焦镜；5.75光纤；5.76散射片；5.77光阑；5.78望远系统。
具体实施方式
下面参照附图描述本发明的示例性实施方式。应当理解，这些具体的说明仅用于示教本领域技术人员如何实施本发明，而不用于穷举本发明的所有可行的方式，也不用于限制本发明的范围。
若非特殊说明，以图1、图3和图4所示的空间来定义根据本发明的芯片及芯片组件中的“上”和“下”的方位。
(芯片组件)
首先参照图1至图4介绍根据本发明的芯片组件的基本结构。
根据本发明的芯片组件包括基板10、多个(图1中为八个)芯片20(即微全分析芯片)和存储器60。
基板10的上表面设有凹陷的芯片安装槽101用于安装芯片20，应当理解，图1中只示意性地示出了芯片安装槽101的位置，并未按比例关系显示芯片安装槽101的形状和尺寸。每一个芯片安装槽101用于安装一个芯片20。
基板10的内部具有多组基板微流道，芯片20的内部具有芯片微流道(下文将结合图5和图6进一 步说明芯片微流道的具体结构)，每一组基板微流道与一个芯片20的芯片微流道相通，使细胞、分子标签(也简称为标签)和细胞隔离介质(也简称为隔离介质)能够在基板微流道和芯片微流道之间流通。
每组基板微流道包括上游基板微流道和下游基板微流道，芯片20连接于上游基板微流道和下游基板微流道之间。
每一个上游基板微流道具有开放于基板10的上表面的两个或三个入口。例如，图4示出了基板10的上表面具有两个入口的方案，其中一个入口用于使隔离介质进入基板微流道，另一个入口用于使细胞和分子标签载流液进入基板微流道。又例如，图1示出了基板10的上表面具有三个入口的方案，这三个入口分别供隔离介质、细胞悬浮液和分子标签进入基板微流道。
首先参照图1至图3介绍基板10的上表面具有三个入口的实施方式。在该实施方式中，每一个供细胞悬浮液进入的入口与一个细胞加样杯31相通，每一个供隔离介质进入的入口与一个隔离介质加样杯41相通，每一个供分子标签进入的入口与一个标签加样杯51相通。通过上述三种加样杯，可以一次将较大剂量的细胞、隔离介质和分子标签加入芯片中进行处理。加样的方式可以是作业者手动加样，或是通过仪器自动加样。
加样杯在与上游基板微流道连通处设有导流口，使细胞悬浮液或分子标签或隔离介质在气路模块(图未示)的控制下流入微流道。
细胞、分子标签和隔离介质在流经芯片微流道后，一个分子标签与一个细胞结合成一个带标签的细胞，此时的溶液被称为细胞和分子标签载流液；之后具有连续液相的细胞和分子标签载流液被隔离介质剪切为分散液相的细胞和分子标签载流液，即单细胞被分散隔离，获得单细胞样本。
优选地，为每个细胞加样杯31设置细胞加样杯盖32，为每个隔离介质加样杯41设置隔离介质加样杯盖42，为每个标签加样杯51设置标签加样杯盖52。
对于图4所示的基板10的上表面具有两个入口的实施方式，每一个供隔离介质进入的入口与一个隔离介质加样杯41相通，每一个供细胞和分子标签载流液进入的入口与一个细胞和标签加样杯531相通。细胞和标签加样杯531内同时容纳有细胞悬浮液与分子标签。优选地，为每个细胞和标签加样杯531设置细胞和标签加样杯盖532。
每一个下游基板微流道具有在基板10的下表面朝下方开口的出样口103，完成标记和隔离的单细胞样本能够在重力作用下从出样口103流出。依靠重力作用获取的单细胞样本，比借助其他工具获取的单细胞样本更不容易发生损坏，所获取的单细胞样本质量更高。
优选地，芯片组件还包括例如呈试管状的收集容器70(参见图4)，收集容器70与基板10通过支架连接，使得从出样口103流出的单细胞样本能流入收集容器70。优选地，收集容器70为透明的容器，以利于下文将介绍的通过光场作用使分子标签脱离载体的操作。
优选地，为加强基板10的密封效果，基板10的下表面依次设有基板下封片104和下密封垫105。
应当理解，一个芯片组件所包含的多个芯片20可以是不同的，即这些芯片20可以具有不同的芯片微流道，从而使得一个芯片组件可以用于处理多个不同的样本；即使是多个同样的芯片20，也可以根据需要用于处理不同的样本。因此，将多个芯片20集成于一个基板10的方式增加了芯片组件对于样本的处理能力。
另一方面，设置于基板10的基板微流道可以是相同的，当需要不同的芯片20时，只需要在芯片组件的生产线上为同一个规格的基板10安装不同的芯片20即可。这种模块化的生产组织方式，大大节约了生产制造成本。
由于同样的基板10可以安装不同的芯片20构成不同的芯片组件，每一个芯片组件又可以处理多个不同的样本，为有效管理芯片组件，优选地，每一个芯片组件所包含的多个芯片20的信息被提前记录在存储器60中。优选地，基板10具有存储器安装槽102以安装存储器60。
优选地，存储器60为具有可读写功能的电子存储器。例如，存储器60可以是电子可擦除存储器(EEPROM)、闪存存储器(FLASH)、铁电存储器(FRAM)或固态硬盘(SSD)。
当使用某一芯片组件时，可以借助电子设备读取存储器60内的信息而获知该芯片组件所包括的每一个芯片20的信息。且在样本处理过程中，可以借助电子设备将新的信息写入存储器60中，从而方便记录每一个芯片20的使用情况。例如，一个芯片组件可以用于多次样本处理试验，每一次样本处理试验使用该芯片组件中的一部分未被使用过的芯片20，从而保证每一个芯片20不被重复使用。
(微全分析芯片)
接下来参照图5和图6介绍根据本发明的芯片20的微流道的结构。
图5示出了对应于上游基板微流道具有三个入口的芯片20的微流道的结构。在该实施方式中，芯片20的微流道包括三个分别与上游基板微流道的三个入口相对应的流入口，即细胞口11、标签口12和隔离介质口13，芯片微流道还包括一个用于连接到下游基板微流道的流出口14。流入口位于芯片20的上表面，流出口14位于芯片20的下表面。
各分支流道将上述三个流入口和一个流出口连接在一起。在本实施方式中，从细胞口11出发，分出两个支路的对称布置的细胞微流道f1。每个细胞微流道f1曲折地布置，使得流道的流阻在细胞微流道f1的流动路径上具有变化，细胞悬浮液的液相流速将随流阻而改变。由于不同大小的细胞在制备成悬浮液时，其沉降速率不尽相同，当细胞悬浮液流至细胞微流道f1时，细胞在液相中的分散程度也不相同，而细胞微流道f1在流动路径上的流阻变化使得分散不均的细胞可以在液相载流的调节下逐渐分散均匀，避免细胞集聚或结团。图5中的箭头a示出了细胞流向。
从标签口12出发，形成有标签微流道f2。优选地，标签微流道f2的长度小于细胞微流道f1的长度。标签微流道f2位于两个细胞微流道f1之间，并与两个细胞微流道f1交汇于第一汇合口C1。优选地，在第一汇合口C1处，标签微流道f2与两个细胞微流道f1的夹角均为90°。当标签微流道f2与细胞微流道f1汇合后，分子标签与细胞结合在一起，使悬浮液内的细胞成为带标签的细胞，液体成为细胞和分子标签载流液。图5中的箭头b示出了标签流向。
从第一汇合口C1出发，向下游延伸有标签细胞微流道f4，用于使细胞和分子标签载流液进一步向下游流动、等待隔离介质的加入。
从隔离介质口13出发，分出两个支路的对称布置的隔离介质微流道f3。隔离介质微流道f3朝第一汇合口C1延伸、并与标签细胞微流道f4交汇于第一汇合口C1下游的第二汇合口C2。优选地，标签细胞微流道f4位于两个隔离介质微流道f3之间，在第二汇合口C2处，标签细胞微流道f4与两个隔离介质微流道f3的夹角均为90°。从而，隔离介质以垂直切入的方式，将带标签的细胞逐个隔离开，使具有连续液相的载流液变成分散液相的载流液，得到单细胞样本。图5中的箭头c示出了隔离介质流向。
从第二汇合口C2出发，向下游延伸有单细胞微流道f5，单细胞微流道f5连通至流出口14，从而单细胞样本能够被进一步输送至出样口103(参见图1、图3和图4)。图5中的箭头d示出了单细胞样本流向。
优选地，在第一汇合口C1和第二汇合口C2处的微流道的内径小于其它路径处的微流道的内径，也称在第一汇合口C1和第二汇合口C2处微流道形成缩口。缩口使得各微流道在交汇使流道内的细胞在被加标签或被隔离的过程中，能逐个进行。
优选地，单细胞微流道f5的内径大于芯片20的其它部分的微流道的内径。
应当理解，虽然在图5中，细胞口11、细胞微流道f1、标签口12和标签微流道f2位于一侧，而隔离介质口13和隔离介质微流道f3位于另一侧，但是这并不是必须的，例如，隔离介质口13和隔离介质微流道f3也可以和细胞口11位于同一侧。
应当理解，在一个芯片20内，细胞微流道f1和隔离介质微流道f3的数量可以不限于是两个，例如，细胞微流道f1也可以只有一个。本发明对细胞口11、标签口12、隔离介质口13、流出口14、细胞微流道f1、标签微流道f2、隔离介质微流道f3、标签细胞微流道f4和单细胞微流道f5的具体布置位置和形状不作限制。
图6示出了对应于上游基板微流道具有两个入口的芯片20的微流道的一个实施方式的结构。在该实施方式中，芯片20的微流道包括两个分别与上游基板微流道的两个入口相对应的流入口，即细胞与标签口111和隔离介质口13，芯片微流道还包括一个用于连接到下游基板微流道的流出口14。流入口位于芯片20的上表面，流出口14位于芯片20的下表面。该实施方式中，细胞与分子标签被混合在一起提前完成分子标签与细胞的结合，即进入细胞与标签口111的已经是细胞和分子标签载流液。
细胞与标签口111与第一标签细胞微流道f11相连，将带标签的细胞向下游输送。第一标签细胞微流道f11的下游端连接缓冲区C0，缓冲区C0内设有多个障碍物(例如微小的柱状的障碍物)，使细胞和分子标签载流液在经过缓冲区C0时，带标签的各细胞能被障碍物分散开而均匀分布。
缓冲区C0的下游连接若干(图6中为三个)第二标签细胞微流道f12，优选地，第二标签细胞微流道f12的内径小于第一标签细胞微流道f11的内径，并使带标签的细胞在流过第二标签细胞微流道f12时在微流道的横截面上是单个通过的。图6中的箭头e示出了细胞和分子标签载流液流向。
多个第二标签细胞微流道f12首先交汇于第一汇合口C1，在第一汇合口C1的下游形成第三标签细胞微流道f13，第三标签细胞微流道f13将在下游与隔离介质交汇。应当理解，第三标签细胞微流道f13的长度可以非常短。
从隔离介质口13出发，分出两个支路的对称布置的隔离介质微流道f3。隔离介质微流道f3朝第一汇合口C1延伸、并与第三标签细胞微流道f13交汇于第一汇合口C1下游的第二汇合口C2。优选地，第三标签细胞微流道f13位于两个隔离介质微流道f3之间。隔离介质从细胞流动方向的侧方切入，将带标签的细胞逐个隔离开，形成单细胞样本。图6中的箭头c示出了隔离介质流向。
从第二汇合口C2出发，向下游延伸有单细胞微流道f5，单细胞微流道f5连通至流出口14，从而单细胞样本能够被进一步输送至出样口103(参见图1、图3和图4)。图6中的箭头d示出了单细胞样本流向。
(单细胞样本制备方法)
参照图7，简单总结使用根据本发明的微全分析芯片或芯片组件制备单细胞样本的方法，其包括：
(i)准备细胞悬浮液。
(ii)使分子标签与细胞混合，形成细胞和分子标签载流液。
(iii)使细胞隔离介质作用于细胞和分子标签载流液，具有连续液相的载流液在来自隔离介质的剪切力作用下，变成分散液相，单细胞被分散隔离。
(iv)收集被细胞隔离介质分散隔离的单细胞样本。
(单细胞样本制备和处理装置&单细胞样本微滴的处理方法)
流入收集容器70的单细胞样本呈微滴的形式。通常在该微滴(以下也将单细胞样本称为单细胞样本微滴或微滴)中，分子标签以结合于某一载体(例如分子标签承载微球或磁珠)的形式存在。为了方便后续二代测序(Next Generation Sequencing,NGS)建库工作，第一，需要使微滴中的分子标签与载体脱落，以增加标签上的例如poly T与mRNA的poly A尾配对结合的几率；第二，需要打碎微滴(也称为微滴破乳)，使结合细胞mRNA的分析标签混合在一起。
接下来，参照图4、图8和图9介绍根据本发明的实现使分子标签与载体脱落的方法和微滴破乳的方法，以及能实现上述整个处理过程的单细胞样本制备与处理装置。
参照图9，根据本发明的单细胞样本制备与处理装置包括电气控制系统1、芯片组件2、电源3、操作显示单元4和多个功能模块(包括芯片加载模块5.1、传感器模块5.2、加热和制冷模块5.3、电极模块5.4、气路模块5.5、液路模块5.6和光路模块5.7)。
电气控制系统1与电源3相连，其包括多个子单元，用于控制各功能模块的工作。
具体地，电气控制系统1包括专用计算机1.1、运动控制单元1.2、样本检测单元1.3、温度控制单元1.4、逆变高压发生单元1.5、气路控制单元1.6、液路控制单元1.7和光场发生单元1.8。
专用计算机1.1与操作显示单元4电连接，用于可视化地输入或输出指令。
运动控制单元1.2用于控制芯片加载模块5.1。芯片加载模块5.1例如包括电机、运动连杆和支架H(同时参照图8)，作业者将装载有芯片20和各加样杯的基板10置于芯片加载模块5.1的支架H上，支架H能够在运动控制单元1.2的控制下运动到指定工作位置、使基板10位于收集容器70的上方，以保证后续处置操作的精确开展。
样本检测单元1.3用于接受来自传感器模块5.2的信号，从而为系统提供控制参数。传感器模块5.2可以包括流速传感器、压力传感器和液位传感器等。流速传感器用于测量流过芯片组件的各微流道的液体的流速，压力传感器和液位传感器分别用于检测各容器(例如包括细胞加样杯31、隔离介质加样杯41、标签加样杯51、细胞和标签加样杯531和收集容器70)内的压力和液位。
温度控制单元1.4用于控制加热和制冷模块5.3所提供的温度，从而为芯片组件2所在的工作位置提供合适的反应温度。
气路控制单元1.6用于控制气路模块5.5所提供的气体的量，从而使细胞加样杯31、隔离介质加样杯41、标签加样杯51或细胞和标签加样杯531内的液体能更顺畅地流入芯片组件2的微流道。
液路控制单元1.7用于控制液路模块5.6。液路模块5.6例如用于向隔离介质加样杯41内自动添加隔离介质，简化了作业者的操作。
光场发生单元1.8用于控制光路模块5.7，以给芯片组件2的收集容器70提供合适能量的光场，从而使收集容器70内的单细胞样本的分子标签脱离其载体。
光路模块5.7例如可以参照图10所示，其包括光源5.71、准直镜5.72、反射镜5.73、聚焦镜5.74、光纤5.75、散射片5.76、光阑5.77和望远系统5.78。
光源5.71例如为LED光源、激光光源、紫外光源或汞灯。光源5.71产生光波的波长峰值优选地在254nm至450nm之间。
光源5.71发出的光依次透过准直镜5.72、反射镜5.73、聚焦镜5.74、光纤5.75、散射片5.76、光阑 5.77和望远系统5.78而照射到收集容器70。通过控制光阑5.77的孔径大小、以及控制望远系统中各透镜的规格和它们之间的距离，可以控制照射到收集容器70的光斑大小和光能量的大小。
经过光场照射后的单细胞样本微滴，其中的分子标签将从载体上脱离，脱离的分子标签游离于液体中，增加标签上的poly T与mRNA的poly A尾配对结合的几率。
在光场作用一定时间使分子标签脱离载体后，逆变高压发生单元1.5将控制电极模块5.4而给收集容器70内的微滴施加电场。
接下来继续参照图4介绍电极模块5.4是如何工作而实现“微滴破乳”的。
在收集容器70的外周设置第一电极81和第二电极82，第一电极81和第二电极82在竖直方向上间隔开，使得第一电极81和第二电极82之间形成的电场具有在竖直方向上的分量。
在图4所示的实施方式中，每个收集容器70的外周的上部区域均有一个环形的第一电极环810，每个收集容器70的外周的下部区域均有一个筒形的第二电极环820。优选地，第二电极环820的高度等于或大于收集容器70的位于第一电极环810的下方的部分(简称收集容器70的下半部)的高度，从而收集容器70的下半部能被完全收容于第二电极环820的内腔。多个第一电极环810相连而构成第一电极81，多个第二电极环820相连而构成第二电极82。
应当理解，第二电极环820也可以只环绕收集容器70的轴向上的中部区域、而不包围收集容器70的整个下部，此时，环形的第二电极环820例如为具有中孔的板状。
应当理解，图4所示出的是第一电极81和第二电极82的一种优选的实施方式，在其他可能的实施方式中，第一电极81和第二电极82也可以设置成由一个环包围多个收集容器70的形式。
优选地，第一电极81和第二电极82的制作材料包括金属和/或导电非金属，例如，第一电极81和第二电极82的制作材料可以选自石墨、铝、铜中的一者或多者。
逆变高压发生单元1.5连接直流或交流电源，电源的电压范围为1至100V，优选地，逆变高压发生单元1.5的输入电压为24V；第一电极81和第二电极82所产生的电压为-10KV至+10KV的交流脉冲电压，交流脉冲电压的频率和电压值可以根据第一电极81和第二电极82之间的距离以及微滴之间的距离而调整。
电场未作用时，收集容器70内的微滴在重力和载液浮力的作用下受力平衡，在微观上做随机运 动。当第一电极81和第二电极82之间产生电场后，微滴受到电场力而振动、使得维持微滴球体形状的表面张力发生剧烈变化。在电场方向和重力场方向平行的情况下，电场的电子作用力最大，此时微滴的随机运动和受电场振动的运动产生共振，微滴表面张力失去平衡，微滴沿重力场方向发生振幅最大的振动，不平衡的表面张力导致微滴发生融合，即微滴破乳。
液滴打碎后，结合细胞mRNA的分子标签混合到一起，方便后续的NGS建库工作。
此外，在电场作用的过程中，具有较大体积和质量的第二电极82将产生较大的热量，从而充当热源以加热其环绕的收集容器70。因此，在本发明中，分子标签捕获单一细胞的mRNA、并经过逆转录合成cDNA片段后立即被加热，而不需要进行移液、转管的操作。
本发明至少具有以下优点中的一个优点：
(i)使用本发明的微全分析芯片或芯片组件制备的单细胞样本，细胞和细胞之间被隔离介质完全隔离，避免了细胞间的交叉污染。
(ii)单细胞样本依靠重力作用流出微全分析芯片或芯片组件，细胞不容易发生损伤，获得的单细胞样本质量高。
(iii)微全分析芯片采用模块化的设计和制造方式，同样的基板10可以安装不同的芯片20以得到不同规格的芯片组件，芯片组件的制造成本低。
(iv)一个芯片组件包括多个芯片20，使得一个芯片组件可以用于处理多个样本，提高样本的制备效率。
(v)通过存储器60管理一个芯片组件所包括的多个芯片20的信息，能有效且高效地记录各芯片20的使用情况，样本之间不会发生污染。
(vi)根据本发明的单细胞样本制备与处理装置通过施加电场实现微滴破乳，不需要向反应体系增加其它试剂。
(vii)根据本发明的单细胞样本制备与处理装置通过施加光场使分子标签脱离其承载载体，不需要向反应体系增加其它试剂。
(viii)根据本发明的单细胞样本制备与处理装置集成了自动化的控制模块，作业者例如可以不需要手工添加试剂，且试剂的添加量能被精确控制，节约了劳动量及试剂成本。
当然，本发明不限于上述实施方式，本领域技术人员在本发明的教导下可以对本发明的上述实 施方式做出各种变型，而不脱离本发明的范围。例如：
根据本发明的微全分析芯片和芯片组件能够用于处理的细胞包括但不限于：细胞系细胞(如人类HEK293T、小鼠NIH3T3)，组织消化细胞(如小鼠脑组织E18 neuron)，人外周血单核细胞(PBMC)。所处理的细胞直径通常为5um至30um，但本发明对此不作限制。

Claims (13)

  1. 一种单细胞样本制备和处理装置，其包括用于制备单细胞样本微滴的芯片组件(2)和用于使所述单细胞样本微滴破乳的电极模块(5.4)，其中，
    所述芯片组件(2)包括芯片(20)和收集容器(70)，所述收集容器(70)用于收集由所述芯片(20)制备的所述单细胞样本微滴，
    所述电极模块(5.4)包括第一电极(81)和第二电极(82)，所述第一电极(81)和所述第二电极(82)均设置于所述收集容器(70)的外周，且所述第一电极(81)和所述第二电极(82)在竖直方向上间隔开、所述第一电极(81)和所述第二电极(82)在所述竖直方向上至少部分地重合，使得所述第一电极(81)和所述第二电极(82)之间形成的电场具有在竖直方向上的分量。
  2. 根据权利要求1所述的单细胞样本制备和处理装置，其特征在于，所述第一电极(81)位于所述第二电极(82)的上方，
    所述第一电极(81)包括环形的第一电极环(810)，所述收集容器(70)穿过所述第一电极环(810)，
    所述第二电极(82)包括筒形的或环形第二电极环(820)，所述收集容器(70)的下部部分被容纳于所述第二电极环(820)的内腔。
  3. 根据权利要求2所述的单细胞样本制备和处理装置，其特征在于，所述收集容器(70)有N个，N大于或等于2，
    所述第一电极环(810)有N个，所述第二电极环(820)有N个，
    每个所述收集容器(70)的外周设有一个所述第一电极环(810)和一个所述第二电极环(820)，
    N个所述第一电极环(810)连接在一起，N个所述第二电极环(820)连接在一起。
  4. 根据权利要求1所述的单细胞样本制备和处理装置，其特征在于，所述第一电极(81)和所述第二电极(82)上加载的电压为在-10KV至+10KV之间的交流脉冲电压。
  5. 根据权利要求1所述的单细胞样本制备和处理装置，其特征在于，所述装置还包括逆变高压发生单元(1.5)，所述第一电极(81)和所述第二电极(82)所加载的电压的大小和频率受所述逆变高压发生单元(1.5)的控制，所述逆变高压发生单元(1.5)连接直流或交流电源，所述电源的电压范围为1至100V。
  6. 根据权利要求1所述的单细胞样本制备和处理装置，其特征在于，所述第二电极(82)的制作材料包括石墨、铝和铜中的一者或多者。
  7. 根据权利要求1所述的单细胞样本制备和处理装置，其特征在于，所述装置还包括光路模块(5.7)，所述光路模块(5.7)用于提供照射所述收集容器(70)的光波，所述光波用于使所述单细胞样本微滴内的分子标签脱离用于承载所述分子标签的载体。
  8. 根据权利要求7所述的单细胞样本制备和处理装置，其特征在于，所述光波的波长在254nm至450nm之间。
  9. 根据权利要求7所述的单细胞样本制备和处理装置，其特征在于，所述光路模块(5.7)包括光阑(5.77)和望远系统(5.78)，所述光阑(5.77)的孔径大小能够被调节，所述望远系统(5.78)的各透镜之间的距离能够被调节。
  10. 根据权利要求1至9中任一项所述的单细胞样本制备和处理装置，其特征在于，所述装置还包括液路模块(5.6)，所述液路模块(5.6)用于根据预定程序向所述芯片组件(2)添加试剂。
  11. 根据权利要求1至9中任一项所述的单细胞样本制备和处理装置，其特征在于，所述装置还包括传感器模块(5.2)，所述传感器模块(5.2)包括以下各传感器中的一者或多者：
    流速传感器，用于测量流过所述芯片(20)的微流道的液体的流速；
    压力传感器，用于测量所述芯片组件(2)的各容器内的压力；
    液位传感器，用于测量所述芯片组件(2)的各容器内的液体的液位。
  12. 一种单细胞样本微滴的处理方法，其特征在于，使用根据权利要求1至11中任一项所述的装置对所述单细胞样本微滴进行制备和处理，所述单细胞样本微滴收容于所述收集容器(70)内，所述方法包括：
    利用所述电极模块(5.4)向所述收集容器(70)施加具有在竖直方向上的电场分量的电场，使所述单细胞样本微滴受电场作用发生表面张力的变化而破碎，从而所述单细胞样本微滴内的分子标签游离于单细胞样本中，以便于捕获细胞中的mRNA或DNA。
  13. 一种单细胞样本微滴的处理方法，其特征在于，使用根据权利要求1至11中任一项所述的装置对所述单细胞样本微滴进行制备和处理，所述单细胞样本微滴收容于所述收集容器(70)内，所述 方法包括：
    向所述收集容器(70)内的所述单细胞样本微滴照射光波，使所述单细胞样本微滴内的分子标签脱离所述分子标签的载体。
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