WO2023173408A1 - 微量液滴内功能细胞低损耗单细胞测序文库构建方法和装置 - Google Patents

微量液滴内功能细胞低损耗单细胞测序文库构建方法和装置 Download PDF

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WO2023173408A1
WO2023173408A1 PCT/CN2022/081698 CN2022081698W WO2023173408A1 WO 2023173408 A1 WO2023173408 A1 WO 2023173408A1 CN 2022081698 W CN2022081698 W CN 2022081698W WO 2023173408 A1 WO2023173408 A1 WO 2023173408A1
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droplet
droplets
injection
inlet
chip
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PCT/CN2022/081698
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English (en)
French (fr)
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刘亚
王诗雨
刘杨
胡定龙
高开
吕孟华
刘龙奇
顾颖
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深圳华大生命科学研究院
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Priority to PCT/CN2022/081698 priority Critical patent/WO2023173408A1/zh
Publication of WO2023173408A1 publication Critical patent/WO2023173408A1/zh

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    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to a method and device for constructing a low-loss single-cell sequencing library of functional cells in micro-droplets.
  • Droplet microfluidics can quickly package single cells in a micro-volume to achieve high-throughput cell function testing at the single-cell level, which has significant advantages and application development prospects.
  • Microdroplet screening technology based on fluorescence excitation (FADS) is an effective technology for detecting biochemical reactions within microdroplets and screening target droplets. It is one of the most promising alternative technologies after flow cytometry. Compared with flow cell sorting technology, droplet sorting technology can quantitatively analyze cell secretions, cell-cell interactions, etc. without damaging cells. It has good compatibility with single-cell sequencing technology and can meet the needs of Important industrial needs such as antibody screening, tumor immunotherapy cell preparation, and industrial bacteria screening have a wide range of commercial application scenarios.
  • the existing technical routes mainly include: 1) Capturing one droplet at a time through the size of the micropores on the microwell array, and then adding the droplets containing the detection reagent to realize the addition of the detection reagent; 2) Using high-precision liquid flow control , realize the electrical fusion of the front and rear droplets, and complete the addition of detection reagents to the cell-containing droplets.
  • the paper "Electro-Coalescence of Digitally Controlled Droplets” introduces a method of designing a deceleration structure on a PDMS chip and ordering the fusion of droplets arriving at the deceleration structure under the action of an electric field (as shown in Figure 1).
  • one droplet can contain cells to be detected, and another droplet can contain detection reagents.
  • cell lysis detection in the droplets can be achieved.
  • the limitation of this technology is that it needs to ensure that the droplet parcels arriving successively are different, and the deceleration structure design needs to match the droplet flow rate.
  • the US2012/0258487A1 patent states a method similar to that described in technology 1).
  • Different droplet channels are used to prepare droplets to wrap different plasmid assemblies (Gene1, Gene2, Gene3, Vector, etc.), and the sequence of droplets arriving at the intersection is controlled through high-precision liquid flow, for example: making a Gene1 droplet arrive at the intersection Then, a Gene2 droplet arrived immediately, and the two droplets were ordered to fuse under the action of the electric field, achieving sequential control of component assembly.
  • the advantages and disadvantages of this technology are similar to technology 1), but the difference is that due to the lack of a deceleration structure, it is more difficult to control droplet fusion, which puts higher requirements on the stability of the liquid flow.
  • Patent CN110650802A introduces a method of using ethyl orthosilicate to prepare a microfluidic chip.
  • the uses include a method for detecting proteins secreted by immune cells: 1) prepare droplets containing cells to be detected and capture microbeads; 2) conduct droplets Screening; 3) Design an ethyl orthosilicate array chip, use micropores to capture sorting droplets, and then add droplets containing sequencing reagents above the sorting droplets and fuse them to achieve single-cell grouping under droplet-free demulsification conditions. Learning information detection. This strategy avoids the loss of small numbers of cells during droplet demulsification.
  • droplet capture based on micropores puts forward higher requirements for the stability of droplet size; the entire array of chips is difficult to process and requires multi-layer bonding, making it difficult to industrialize production; droplet fusion requires the use of equipment such as an electric iron. , the price is high.
  • the invention provides a method and device for constructing a low-loss single-cell sequencing library of functional cells in micro-droplets.
  • the technical problems to be solved by the present invention are: 1) reducing the low cell recovery rate caused by the demulsification of droplets after sorting, making it impossible to perform single-cell library construction and sequencing; 2) reducing the high-precision flow control device of the droplet fusion step Cost; 3) Achieve single cell library construction with trace amounts of cells in droplets; 4) Simplify the operating steps.
  • the invention provides a droplet collection device, which includes an inner tube, an outer tube and a first negative pressure device.
  • the upper end of the inner tube is connected to the droplets to be collected, and the lower end is connected to the outer tube; the upper end of the outer tube is open.
  • the inner tube is sleeved in the outer tube; the outer tube is connected with the first negative pressure device.
  • the inner tube is an inverted tapered tube with a larger top and a smaller bottom.
  • the lower end outlet of the tapered tube is less than 1 mm and greater than 200 microns.
  • the first negative pressure device is a syringe.
  • the invention also provides a droplet injection device, which includes a droplet injection chip.
  • the droplet injection chip is provided with a droplet generation oil inlet, a droplet inlet, an injection reagent inlet, a droplet outlet, a main flow channel and a micro flow channel. ;
  • the droplet inlet and the droplet outlet are respectively located at both ends of the main channel, and the droplet generation oil inlet and the injection reagent inlet are connected to the main channel through the microfluidic channel; the injection reagent inlet and the main channel are connected with electrodes; so
  • the droplets contact the droplets sequentially in the main channel to generate oil and injection reagent; a droplet wrapping the injection reagent is formed at the outlet of the droplet.
  • the droplet inlet of the droplet injection device chip is connected to the droplet collection device; the droplet outlet is connected to the droplet collection tube, and the droplet collection tube is connected to the second negative pressure device.
  • the second negative pressure device is a syringe.
  • the droplets contain magnetic beads, and one of the width and height of the main channel is greater than 2 times the diameter of the magnetic beads.
  • One of the width and height of the main channel is smaller than the droplet diameter.
  • the invention also provides a device for constructing a single-cell sequencing library in micro-droplets, which includes a droplet generation chip and a droplet injection device.
  • the droplets generated by the droplet generation chip are connected to the droplet inlet of the droplet injection device.
  • the droplet generation chip also includes a droplet sorting chip.
  • the droplets generated by the droplet generation chip are connected to the upper end of the inner tube of the droplet collection device through the droplet sorting chip, and are collected by the droplet collection device. Finally, it is connected with the droplet inlet of the droplet injection device.
  • the present invention also provides a method for constructing a single-cell sequencing library in micro-droplets, using the single-cell sequencing library construction device in micro-droplets described in 0.
  • step 1 a sorting and collection step is included between step 1 and step 2, as follows:
  • the sorted droplets enter the inner tube through the upper inlet of the inner tube of the droplet collection device. As the oil and droplets flow into the inner tube, the droplets are suspended in the upper layer. The oil flows through the bottom of the inner tube to the outer tube. When the liquid The droplets float to the inner tube opening, and the syringe is pulled to extract the oil from the outer tube so that the liquid level in the inner tube drops;
  • Figure 1 shows a droplet fusion device based on a deceleration structure.
  • Figure 2 shows a droplet fusion device based on high-precision liquid flow control.
  • Figure 3 is a schematic diagram of the entire process of droplet generation-sorting-injection.
  • Figure 4 is a three-dimensional view of the droplet injection device.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are all conventional methods unless otherwise specified.
  • the test materials used in the following examples were all purchased from conventional biochemical reagent stores unless otherwise specified.
  • the quantitative experiments in the following examples were repeated three times, and the results were averaged.
  • the present invention provides a droplet collection device, including an inner tube 401, an outer tube 402 and a first negative pressure device 403.
  • the upper end of the inner tube 401 is connected to the droplets to be collected, and the lower end is connected to the outer tube.
  • the tubes 402 are connected; the upper end of the outer tube 402 is open, and the inner tube 401 is sleeved in the outer tube 402; the outer tube 402 is connected with the first negative pressure device 403.
  • the inner tube 401 is an inverted tapered tube with a larger top and a smaller bottom.
  • the outlet at the lower end of the tapered tube is less than 1 mm and greater than 200 microns.
  • the first negative pressure device 403 is a syringe, and the outer tube 402 and the first negative pressure device 403 are connected through an oil drain pipe 404 .
  • the upper end of the inner tube 401 is connected with the liquid droplets to be collected through a droplet conduit 405 .
  • the present invention also provides a droplet injection device, including a droplet injection chip.
  • the droplet injection chip is provided with a droplet generation oil inlet 301, a droplet inlet 302, and an injection reagent inlet. 303.
  • Droplet outlet 305, main channel 308 and microfluidic channel 309; the droplet inlet 302 and the droplet outlet 305 are located at both ends of the main channel respectively, and the droplet generating oil inlet 301 and the injection reagent inlet 303 pass through the microfluidic channel.
  • an electrode 304 is provided at the connection between the injection reagent inlet 303 and the main channel 308; the droplets contact the droplets sequentially in the main channel 308 to generate oil and injection reagent; a wrapped injection reagent is formed at the droplet outlet. droplets.
  • the droplet inlet of the droplet injection device chip is connected to the droplet collection device; the droplet outlet 305 is connected to the droplet collection tube 306 , and the droplet collection tube 306 is connected to the second negative pressure device 307 .
  • the second negative pressure device 306 is a syringe.
  • the droplets contain magnetic beads, and one of the width and height of the main channel 308 is greater than 2 times the diameter of the magnetic beads.
  • One of the width and height of the main channel 308 is smaller than the droplet diameter.
  • the invention provides a device for constructing a single cell sequencing library in micro-droplets, which includes a droplet generation chip and a droplet injection device.
  • the droplets generated by the droplet generation chip are connected to the droplet inlet of the droplet injection device.
  • the droplet generation chip also includes a droplet sorting chip.
  • the droplets generated by the droplet generation chip are connected to the upper end of the inner tube of the droplet collection device through the droplet sorting chip, and pass through the droplet collection device. After collection, it is connected to the droplet inlet of the droplet injection device.
  • the droplet generation device is shown in Figure 3A.
  • 102 and 103 are liquid filling ports for adding cell suspension and other hydrophilic solutions.
  • 101 is a filling port for adding droplets to generate oil.
  • Liquid filling ports 102/103 and filling port 101 are in the micro.
  • the function of the flow channel is that after converging at the intersection point 107, it continues to communicate with the liquid outlet 104 through the micro-channel.
  • the outlet is connected to the generation tube 105 through a sealed conduit, and the generation tube 105 is connected to the syringe 106;
  • the liquid filling ports 102 and 103 are connected to the liquid outlet 104 through a Y-shaped tube; the liquid droplet generating device is a negative pressure driven liquid droplet generating device.
  • the interior of the device is sealed, so that a negative pressure can be formed therein.
  • droplets are added to the filling port 101 to generate oil
  • cell suspension is added to the liquid filling port 102
  • other aqueous reagents such as cell culture fluid are added to the liquid filling port 103.
  • the sealing process of the generation tube can be modified from the cell cryopreservation tube, opening the top of the cryopreservation tube cover, connecting the catheter, and then using hot melt glue to seal the gap between the catheter and the tube cover.
  • the cryopreservation tube cap is tightened, the collection tube is only connected to the outside through the conduit.
  • the syringe 106 is pulled, the pressure inside the device decreases.
  • droplets 101, 102, and 103 continue to enter the device pipeline.
  • the horizontal oil cuts the converged liquid flows of 102 and 103 to form droplets, completing the liquid flow.
  • Droplets are generated and enter the generation tube 105 through the conduit. After the generation of droplets is completed, the pipeline is cut and the droplets in the generation tube 105 are taken out.
  • a cellular genetic material capture carrier needs to be added to the cell suspension.
  • magnetic beads with a diameter of about 20 microns are required.
  • the carrier diameter should be avoided to exceed 30 microns, which will affect the droplet injection. Chip operation.
  • the droplet sorting device is shown in Figure 3B.
  • the droplet sorting device includes: a sample injecting component and a droplet sorting component.
  • a droplet inlet 202 and an oil phase inlet 201 are designed in the sample injection component.
  • the droplet sorting component at least includes a first channel 206, a second channel 204, and a third channel 203, and the first channel, the second channel, and the third channel are arranged in a horizontal "Y" shape.
  • the inventor makes use of the difference in flow velocity ratio on both sides of the "Y"-shaped pipe to make the droplets move to the second channel without the action of external force.
  • metals such as tin are used to prepare electrodes.
  • the positive droplet passes through the first channel, it will emit light under laser excitation.
  • the light signal generated by the droplet can generate a deflection force. This deflection force will make the droplet move to the side of the third channel, thereby achieving target detection. sorting.
  • the droplets collected in the generation tube 105 are transferred to a 1 ml syringe and connected to the sorting droplet inlet 202 via a conduit.
  • the droplet generation oil is put into a 10 ml syringe and connected to the sorting oil phase inlet 201 via a conduit. Place the syringe on the syringe pump, and after starting the syringe pump, the oil injected into the sorting oil phase inlet 201 disperses and accelerates the droplets injected into the sorting droplet inlet 202.
  • the droplets are detected, and the target droplets are sorted by the electrodes and enter the third sorting channel 203, otherwise the droplets flow into the second channel 204.
  • the liquid outlet of the third sorting channel 203 can be connected to a conduit to a collection device, and the collected droplets can be recovered and subjected to single cell sequencing after being destroyed by the droplets.
  • the droplet collection device is composed of an inner tube 401 and an outer tube 402.
  • the inner tube 401 has a tapered structure and the outer tube 402 has a U-shaped bottom.
  • the inner tube 401 is placed inside the outer tube 402, and the inner tube 401 contains liquid It is connected to the outer tube 402 through the bottom to form a connector, and an oil drain pipe 404 connected to the syringe is placed in the outer tube; the liquid outlet of the third channel 203 in Figure 3B is connected to the inner tube 401 through the conduit, and the oil and liquid droplets flow into the inner tube 401 , the droplets are suspended in the upper layer, and the oil flows to the outer tube 402 through the bottom of the inner tube 401.
  • the syringe When the droplets float to the mouth of the inner tube 401, the syringe is pulled to extract the oil in the outer tube 402, causing the liquid level in the inner tube 401 to drop. After completing the droplet collection, take out the inner tube and immediately insert it into the droplet inlet 302 in Figure 3D.
  • the diameter of the bottom of the inner tube 401 is slightly larger than the diameter of the droplet inlet 302, so that the inner tube and the droplet inlet 302 are closely connected; the inner tube is tapered, which makes the droplets more concentrated during their descent and reduces the loss of droplets hanging on the wall; There are no strict requirements for the liquid level of the tube, as long as the liquid droplets do not overflow.
  • the droplet injection device includes a droplet generation oil inlet 301; a droplet inlet 302; an injection reagent inlet 303; a droplet outlet 305 of the chip; an electrode 304; a droplet collection tube 306, a microfluidic channel 309 and a main flow channel. 308.
  • the droplet inlet 302 and the droplet outlet 305 are respectively located at both ends of the main channel.
  • the droplet generation oil inlet 301 and the injection reagent inlet 303 are connected to the main channel 308 through the microfluidic channel 309; the injection reagent inlet 303 is connected to the main channel 308.
  • An electrode 304 is provided at the connection point; the droplets contact the droplets sequentially in the main channel 308 to generate oil and injection reagent; a droplet wrapping the injection reagent is formed at the droplet outlet.
  • the droplet inlet of the droplet injection device chip is connected to the droplet collection device; the droplet outlet 305 is connected to the droplet collection tube 306, and the droplet collection tube 306 is connected to the second negative pressure device 307. Pass.
  • the chip of this device is prepared and bonded by a single layer of PDMS glue, which has mature technology, simple operation and low cost; the liquid power is provided by the negative pressure formed by the syringe, the liquid flow is stable and the equipment cost is low; when used, the pipette tip can be transformed into The tapered tube can realize droplet loading of at least 10 microliters, overcome the droplet loss caused by the dead volume in the syringe pump and chip connection tube, and achieve non-destructive or low-loss loading of micro droplets.
  • This chip has requirements for the height and width of the flow channel. Since hard magnetic beads may be used as the enrichment carrier for cell genetic material in actual operation, the width and height of the flow channel need to be more than 2 times the diameter of the magnetic beads respectively.
  • the distance between the ports is too far, resulting in inability to inject.
  • the flow channel height is required to be smaller than the droplet diameter. Therefore, the chip height and width should meet: height/width>2*magnetic bead diameter; at the same time, height/width ⁇ droplet diameter.
  • the injection phase flow rate is affected by pressure and droplet viscosity.
  • the present invention recommends using 0.4 (w/v) Fiocll solution to adjust the injection phase viscosity to adjust the liquid injection volume; use When using the chip shown in the present invention, it is necessary to add 0.4 (mass to volume ratio, w/v) Ficoll solution until the final concentration of Ficoll is greater than 5% (w/v) to prevent the injection phase from infiltrating the PDMS near the injection port.
  • the droplet injection process is shown in the enlarged view of Figure 3D.
  • the droplet passes through the sample addition port, under the action of the electric field, the oil film between the droplet and the injection phase opens instantly.
  • the injection phase enters the droplet driven by atmospheric pressure.
  • the volume of the added liquid is the same as the liquid droplet.
  • the volume of the droplet itself is related; 301 oil participates in controlling the distance between droplets.
  • the flow rate of oil, droplets, injection phase and droplet interval are fixed; therefore, stable liquid injection into droplets can be achieved .
  • This chip needs to be connected to a continuous voltage of more than 150V, and the voltage can be any periodic waveform such as sine wave, square wave, etc.
  • the injected droplets are left at room temperature for 40 minutes to 2 hours to achieve cell lysis, and the genetic material is captured by magnetic beads in the droplets.
  • Single-cell omics libraries can then be constructed according to the standard method of the DNAelab C4 kit.
  • the power source of the present invention is: the chip-collection tube-syringe are connected to form a closed system.
  • the air inside the system is diluted and the pressure is less than the external atmospheric pressure.
  • liquids 301, 302, and 303 enter the system.
  • the injection phase is powered by the pressure difference between the system and the atmosphere; the distance between the droplet and the injection phase is instantly broken under the action of voltage to achieve communication; due to the external pressure, the injection phase liquid forms a unidirectional flow toward the droplet, and
  • the injection volume is determined by the time when the droplets pass through the injection port and the internal and external pressure difference.
  • the variable factors are single and the system is simple and controllable.
  • Example 1 Micro-volume single cell library construction and sequencing based on the present invention
  • This example uses the present invention to recover single-cell transcriptome information of 5,000-10,000 cells from droplets, proving that the method and device can realize single-cell omics analysis of micro-droplets and cells. Specific steps are as follows:
  • NIH3T3 ATCC No.CCL-92
  • 293T cells ATCC No.CRL-3216
  • a special solution 50% RPMI1640 culture medium (Gibco), 30% 0.4 (w/v) Ficoll, 18% fetal calf serum, 1% penicillin-streptomycin solution, 1% F68 surfactant
  • resuspend 500 or 1000 cells per microliter ( ⁇ l);
  • RNA capture magnetic beads from the DNBelab C4 kit, place it on a magnetic stand (12321D, Invitrogen), wait for 2 minutes, and discard the supernatant;
  • this method and device realize the docking of the mainstream single-cell library construction and sequencing method after the cells react in the droplets, and the cell recovery rate and other parameters are consistent with the mainstream method.
  • the parameters are close, indicating the good compatibility of this method with existing methods.
  • this embodiment has the following key improvements compared with previous methods:
  • Example 2 Single-cell library construction and sequencing of microdroplets after sorting
  • RNA capture magnetic beads from the DNBelab C4 kit, place it on a magnetic stand (12321D, Invitrogen), wait for 2 minutes, and discard the supernatant;
  • step 6 The specific method of droplet sorting in step 6) above is as follows: transfer the droplets to a 1mL syringe, connect it to the inlet 202 via a catheter, and at the same time add the oil phase to the 5mL syringe to connect the inlet 201.
  • the syringes containing the liquid droplets and the oil phase are pushed using syringe pumps respectively, and the liquid droplets flow through the channel 206.
  • the positive droplets will emit light under laser excitation, and the light signal generated by the droplets can generate a deflection force through the electrodes. The deflection force will deflect the droplets toward the 203 channel; while the negative droplets are discarded through the 204 channel.
  • the library cDNA of the present invention is lower than the standard process, but significantly higher than the sorting droplet-emulsification-standardized process library construction, which solves the problem of inability to recover micro-droplet cells (Table 3).
  • Example 3000 mixed cells prepared in Example 1 were used according to the demulsification-cell recovery method. Other parameters were the same as Example 2. The results are shown in Table 3. The cells could not be recovered for single cell library construction.
  • the demulsification-cell recovery method is specific. Includes the following steps:
  • the cells Precipitate the cells; 3) After the droplet generation is completed, there are two layers in the droplet collection tube, the upper layer is water-in-oil droplets, and the lower layer is a small amount of droplets generating oil; use a 1ml syringe to remove the oil in the lower layer of the droplet collection tube; 4) Slowly add 200 microliters of PFO into the droplet collection tube and let it sit for 10 minutes; 5) At this time, there are three layers in the sample collection tube. The lower layer is the oil phase mixture of the oil generated by the droplets and PFO, and the middle layer is the unbroken droplets. The upper layer is the aqueous phase containing cells released by demulsification.
  • the droplet injection chip of this device is prepared and bonded by a single layer of PDMS glue, which has mature technology, simple operation and low cost; the liquid power is provided by the negative pressure formed by the syringe, the liquid flow is stable and the equipment cost is low; when used, a pipette can be used
  • the tip is transformed into a tapered tube, which can realize droplet loading of at least 10 microliters, overcome the droplet loss caused by the dead volume in the syringe pump and chip connection tube, and achieve non-destructive or low-loss loading of micro-droplets.
  • This chip has requirements for the height and width of the flow channel.
  • the width and height of the flow channel need to be more than 2 times the diameter of the magnetic beads respectively.
  • the distance between the ports is too far, resulting in inability to inject.
  • the flow channel height is required to be smaller than the droplet diameter. Therefore, the chip height and width should meet: height/width>2*magnetic bead diameter; at the same time, height/width ⁇ droplet diameter.
  • the injection phase flow rate is affected by pressure and droplet viscosity.
  • the present invention recommends using 0.4 (w/v) Fiocll solution to adjust the injection phase viscosity to adjust the liquid injection volume; use When using the chip shown in the present invention, it is necessary to add 0.4 (mass to volume ratio, w/v) Ficoll solution until the final concentration of Ficoll is greater than 5% (w/v) to prevent the injection phase from infiltrating the PDMS near the injection port.

Abstract

一种微量液滴内单细胞测序文库构建装置及方法，装置包括液滴生成芯片、液滴分选芯片、液滴收集装置和液滴注射装置，液滴生成芯片生成的液滴经过液滴分选芯片与液滴收集装置的内管（401）上端相连通，并经过液滴收集装置的收集后，与液滴注射装置的液滴入口（302）相连通。方法使用微量液滴内单细胞测序文库构建装置。装置成本低；芯片制作工艺简单；使用便捷、可无人值守；兼容不同尺寸液滴；实现液滴内微量细胞的单细胞建库。

Description

微量液滴内功能细胞低损耗单细胞测序文库构建方法和装置 技术领域
本发明涉及微量液滴内功能细胞低损耗单细胞测序文库构建方法和装置。
背景技术
液滴微流控可以在微量体积内对单个细胞快速包裹，实现单细胞水平的高通量细胞功能检测，而具有显著的优势和应用开发前景。基于荧光激发的微液滴筛选技术(FADS)是实现微液滴内生化反应检测和目标液滴筛选的有效技术，是流式细胞仪之后最有潜力的替代技术之一。与流式细胞分选技术相比，液滴分选技术可以在不损伤细胞的前提下对细胞分泌物、细胞间互相作用等进行定量分析，和单细胞测序技术有着良好的兼容性，能够满足抗体筛选、肿瘤免疫治疗细胞制备、工业用菌筛选等重要产业需求，有着广泛的商业应用场景。但是，由于基于液滴的筛选面向高度异质性的筛选对象(细胞、工业菌、细胞互作等)，常常在分选后仅能获取少量液滴，为了进行单细胞精度的组学分析，传统方法需要从液滴内破乳回收细胞，造成大量损失，无法应用于少量液滴的组学分析，阻碍微液滴筛选技术的推广和应用，同时，由于FADS巨大的市场前景，对微量液滴的单细胞建库测序方法有较高的需求。因此，需要开发在不打破液滴的前提下实现液滴内细胞裂解和组学信息捕获的技术。目前已有的技术路线主要包括：1)微孔阵列上通过微孔尺寸实现单次一个液滴捕获，之后加入含有检测试剂的液滴，实现检测试剂的加入；2)借助高精度液流控制，实现前后液滴的电融合，完成检测试剂向含细胞液滴的加入。
“Electro-Coalescence of Digitally Controlled Droplets”论文介绍一种通过在PDMS芯片上设计减速结构，在电场作用下令先后抵达减速结构液滴的融合的方法(如图1所示)。在这种方法中，一种液滴可以包含待检测细胞，另一种液滴可以包含检测试剂，当先后抵达液滴分别包含细胞和检测试剂的时候，可以实现液滴内细胞裂解检测。但是，这种技术的局限是需要保证先后抵达液滴包裹物不同，且减速结构设计需要和液滴流速配合，在实际操作中对液流系统的稳定性提出较高的要求，需要配置高精度的注射泵系统，成本高昂(研究所使用注射泵至少需要3通道，成本约4-5万元左右)。
US2012/0258487A1专利(如图2所示)陈述一种类似于技术1)所述方法。不同的液滴通道制备液滴包裹不同的质粒组装件(Gene1、Gene2、Gene3、Vector等)，通过高精度液流控制液滴抵达交汇口的先后顺序，例如：令一个Gene1液滴抵达交汇口后，一个Gene2液滴立刻抵达，在电场作用下令两个液滴融合，实现组件组装的先后控制。这种技术优缺点与技术1)类似，所不同的是由于缺乏减速结构，液滴融合控制更加困难，对液流的稳定性提出更高的要求。
专利CN110650802A中介绍一种使用正硅酸乙酯制备微流控芯片，用途中包括一种检测免疫细胞分泌蛋白的方法：1)制备液滴包含待检测细胞、捕获微珠；2)进行液滴筛选；3)设计正硅酸乙酯阵列芯片，利用微孔捕获分选液滴，之后在分选液滴上方加入含有测序试剂的液滴并融合，实现液滴免破乳条件下单细胞 组学信息检测。这一策略可以避免液滴破乳过程对少量细胞造成的损失。但是其局限：基于微孔的液滴捕获对液滴尺寸的稳定性提出较高的要求；整列芯片加工难度高，需要多层键合，难以工业化生产；液滴融合需要使用电熨机等设备，价格高昂。
发明公开
本发明提供了一种微量液滴内功能细胞低损耗单细胞测序文库构建方法和装置。
本发明所要解决的技术问题在于：1)减少因为分选后液滴破乳造成的细胞回收率低，无法进行单细胞建库测序；2)降低液滴融合步骤的高精度液流控制装置的成本；3)实现液滴内微量细胞单细胞建库；4)简化操作步骤。
本发明提供一种液滴收集装置，包括内管、外管和第一负压装置，所述内管的上端与待收集液滴相连通，下端与外管相连通；所述外管上端开口，所述内管套设在外管内；所述外管与第一负压装置相连通。
进一步地，所述内管为上大下小的倒锥形管。其中，所述锥形管的下端出口小于1毫米，大于200微米。
进一步地，所述第一负压装置为注射器。
本发明还提供一种液滴注射装置，包括液滴注射芯片，所述液滴注射芯片上设有液滴生成油入口、液滴入口、注射试剂入口、液滴出口、主流道和微流道；所述液滴入口和液滴出口分别位于主流道的两端，液滴生成油入口和注射试剂入口通过微流道与主流道相连通；注射试剂入口与主流道的连通设有电极；所述液滴在主流道内依次接触液滴生成油和注射试剂；在液滴出口处形成包裹注射试剂的液滴。
进一步地，所述液滴注射装置芯片的液滴入口与液滴收集装置相连通；液滴出口与液滴收集管相连通，所述液滴收集管与第二负压装置相连通。
进一步地，所述第二负压装置为为注射器。
进一步地，所述液滴中含有磁珠，所述主流道的宽度和高度中的一种大于2倍磁珠直径。
所述主流道的宽度和高度中的一种小于液滴直径。
本发明还提供一种微量液滴内单细胞测序文库构建装置，包括液滴生成芯片和液滴注射装置，所述液滴生成芯片生成的液滴与液滴注射装置的液滴入口相连通。
进一步地，还包括包括液滴分选芯片，所述所述液滴生成芯片生成的液滴经过液滴分选芯片与液滴收集装置的内管上端相连通，并经过液滴收集装置的收集后，与液滴注射装置的液滴入口相连通。
本发明还提供一种微量液滴内单细胞测序文库构建方法，使用0所述的微量液滴内单细胞测序文库构建装置。
进一步地，包括如下步骤：
1)将待建库细胞、磁珠、液滴生成油加入液滴生成芯片，完成液滴的制备；
2)将制备好的液滴连通至液滴注射装置的液滴入口，并分别在液滴生成油入口和注射试剂入口连通液滴生成油和注射试剂；
3)将液滴注射装置的芯片电极接通电源，打开第二负压装置，收集融合了注射试剂的液滴；
4)将融合了注射试剂的液滴依照DNBelab C4 RNA建库步骤操作。
进一步地，在步骤1和步骤2之间包括分选收集步骤，具体如下：
S1)将步骤1)生成的液滴的使用液滴分选芯片进行分选，得到分选后的混合液滴；
S2)分选后的液滴经液滴收集装置的内管上入口进入内管，随着油和液滴流入内管，液滴悬浮在上层，油经内管底部流动至外管，当液滴上浮至内管口，拉动注射器抽取外管油令内管液面下降；
S3)完成液滴收集后，拿出内管，将插入液滴注射装置的液滴入口，完成液滴注射装置的液滴入口与液滴的连通。
附图说明
图1为基于减速结构的液滴融合装置。
图2为基于高精度液流控制的液滴融合装置。
图3为液滴生成-分选-注射全流程示意图。
图4为液滴注射装置三维图。
本发明的最佳实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
发明概述：
如图3c所示，本发明提供一种液滴收集装置，包括内管401、外管402和第一负压装置403，所述内管401的上端与待收集液滴相连通，下端与外管402相连通；所述外管402上端开口，所述内管401套设在外管402内；所述外管402与第一负压装置403相连通。
所述内管401为上大下小的倒锥形管，所述锥形管的下端出口小于1毫米，大于200微米。
所述第一负压装置403为注射器，所述外管402与第一负压装置403之间通过排油管404连通。所述内管401的上端与待收集液滴通过液滴导管405相连通。
如图3D和图4所示，本发明还提供一种液滴注射装置，包括液滴注射芯片，所述液滴注射芯片上设有液滴生成油入口301、液滴入口302、注射试剂入口303、液滴出口305、主流道308和微流道309；所述液滴入口302和液滴出口305分别位于主流道的两端，液滴生成油入口301和注射试剂入口303通过微流道309与主流道308相连通；注射试剂入口303与主流道308的连通处设有电极304；液滴在主流道308内依次接触液滴生成油和注射试剂；在液滴出口处形成包裹注射试剂的液 滴。
所述液滴注射装置芯片的液滴入口与液滴收集装置相连通；液滴出口305与液滴收集管306相连通，所述液滴收集管306与第二负压装置307相连通。
所述第二负压装置306为为注射器。
所述液滴中含有磁珠，所述主流道308的宽度和高度中的一种大于2倍磁珠直径。
所述主流道308的宽度和高度中的一种小于液滴直径。
本发明提供一种微量液滴内单细胞测序文库构建装置，包括液滴生成芯片和液滴注射装置，所述液滴生成芯片生成的液滴与液滴注射装置的液滴入口相连通。
作为进一步方案，还包括包括液滴分选芯片，所述所述液滴生成芯片生成的液滴经过液滴分选芯片与液滴收集装置的内管上端相连通，并经过液滴收集装置的收集后，与液滴注射装置的液滴入口相连通。
液滴生成装置如图3A所示，102和103为加液口可以加入细胞悬液等亲水溶液，101为加油口用于加入液滴生成油，加液口102/103和加油口101在微流道的的作用在于交汇点107处汇聚后，继续经微流道与出液口104相连通所述处于口通过密闭导管与生成管105连通，所述生成管105与注射器106连通；所述加液口102和103通过呈Y型管连接至出液口104；所述液滴生成装置为负压驱动的液滴生成装置，装置各部件连接后装置内部密闭，从而可以在其中形成负压。使用时，加油口101加入液滴生成油，加液口102加入细胞悬液，加液口103加入其他水相试剂例如细胞培养液。生成管的密闭处理，可以由细胞冻存管改造，在冻存管盖顶部开口，连接导管，再使用热熔胶密闭导管和管盖之间的缝隙。当冻存管盖旋紧时，收集管仅通过和导管和外部连接。当拉动注射器106，装置内压强降低，在大气压驱动下，101、102、103液滴持续进入装置管路，在107处，横向的油切割102、103汇聚的液流，形成液滴，完成液滴生成，并经导管进入生成管105。完成液滴生成后，剪断管路，取出生成管105中的液滴。
使用图3A所示液滴生成装置生成液滴时，需要在细胞悬液中加入细胞遗传物质捕获载体，一般要求为20微米左右直径的磁珠，应该避免载体直径超过30微米，影响液滴注射芯片操作。
所述液滴分选装置如图如图3B所示，液滴分选装置包括：样品进样部件和液滴分选部件。在样品进样部件设计了液滴入口202和油相入口201。液滴分选部件至少包括第一通道206、第二通道204和第三通道203，并且所述第一通道、所述第二通道和所述第三通道呈水平放置“Y”字形排布。在液滴和第二油相分别从液滴入口202和油相入口201进入液滴分选部件后，先流过所述第一通道206，随后根据检测结果流入第二通道204或第三通道203。在本发明中，本发明人利用“Y”形管道两侧流速比的差异，使液滴在无外力作用的情况下，向第二通道移动。在第三通道一侧，使用锡材质等金属制备电极。当阳性液滴通过第一通道时，会在激光激发下发光，液滴由此产生的光信号可以产生偏转力，该偏转力会使液滴向第三通道一侧移动，从而实现对目标的分选。
图3A将生成管105收集的液滴转移至1ml注射器，经导管连接至分选液滴入口202，液滴生成油装入10ml注射器，经导管连接至分选油相入口201。将注射器放置在注射泵上，启动注射泵后，分选油相入口201注入的油分散并加速分选液滴入口202注入的液滴。在第一通道206处，液滴被检测，目标液滴被电极分选进入分选第三通道203，否则液滴流入第二通道204。在分选第三通道203的液 体出口可连接导管至收集装置，收集的液滴经液滴破坏后可回收并进行单细胞测序等。
液滴收集装置如上所述，由内管401和外管402构成，内管401为锥形结构，外管402为U型底，使用时，内管401放置在外管402内，内管401液体经底部和外管402相连，形成连通器，外管内放置一连接注射器的排油管404；图3B第三通道203的液体出口经导管连接至内管401，随着油和液滴流入内管401，液滴悬浮在上层，油经内管401底部流动至外管402，当液滴上浮至内管401口，拉动注射器抽取外管402中的油令内管401液面下降。完成液滴收集后，拿出内管，立即插入图3D的液滴入口302内。此处，内管401底部直径略大于液滴入口302的直径，令内管和液滴入口302紧密相连；内管呈现锥形，令液滴下降过程更加聚集，减少液滴挂壁损失；内管液面无严格要求，满足液滴不溢出即可。
液滴注射装置如图3D所示包括液滴生成油入口301；液滴入口302；注射试剂入口303；芯片的液滴出口305；电极304；液滴收集管306、微流道309与主流道308。所述液滴入口302和液滴出口305分别位于主流道的两端，液滴生成油入口301和注射试剂入口303通过微流道309与主流道308相连通；注射试剂入口303与主流道308的连通处设有电极304；液滴在主流道308内依次接触液滴生成油和注射试剂；在液滴出口处形成包裹注射试剂的液滴。
进一步地，所述液滴注射装置芯片的液滴入口与液滴收集装置相连通；液滴出口305与液滴收集管306相连通，所述液滴收集管306与第二负压装置307相连通。
本装置芯片由单层PDMS胶制备键合，工艺成熟、操作简单、成本低廉；液体动力由注射器形成负压提供，液流稳定、设备成本低廉；使用时，可以使用移液器吸头改造为锥形管，可以实现最少10微升液滴加样，克服注射泵和芯片连接管内死体积造成的液滴损失，实现微量液滴无损或低损失加样。本芯片对流道高度和宽度存在要求，由于实际操作时可能使用硬质磁珠作为细胞遗传物质富集载体，需要流道宽度和高度分别达到磁珠直径2倍以上，但是为了避免液滴和注射口距离过远，导致无法注射，要求流道高度小于液滴直径，因此，芯片高度和宽度应该满足：高度/宽度>2*磁珠直径；同时，高度/宽度<液滴直径。注射相流速收到压强和液滴粘度影响，随着液体粘度增大，注入液滴的体积减少；本发明推荐使用0.4(w/v)Fiocll溶液调节注射相粘度，以调整液体注入体积；使用本发明所示芯片时，需要加入0.4(质量体积比，w/v)Ficoll溶液至Ficoll终浓度大于5％(w/v)，避免注射相浸润注射口附近PDMS。
液滴注射过程如图3D放大图所示，液滴通过加样口时，在电场作用下，液滴和注射相间油膜瞬间打开，注射相在大气压驱动下进入液滴，加入的液体体积和液滴本身体积相关；301的油参与控制液滴间距离，当本装置注射器规格以及拉动距离确定时，油、液滴、注射相流速以及液滴间隔随之固定；因此可以实现液体稳定注入液滴。本芯片需要连接150V以上持续电压，电压可以是正弦波、方波等任意周期波形。
完成注射的液滴室温放置40分钟至2小时，可以实现细胞裂解，遗传物质被液滴内磁珠等捕获，随后可按照DNAelab C4试剂盒标准方法进行单细胞组学建库。
本发明的动力来源为：芯片-收集管-注射器联通形成密闭系统，拉动注射器，系统内部空气稀释，压力小于外部大气压。在外部气压推动下，301、302、303液体进入系统。
注射原理：注射相由系统内和大气压力差提供注射动力；液滴和注射相间隔在电压作用下瞬间打破，实现联通；由于外部压力推动，注射相液体形成向液滴 的单方向流动，且注入量由液滴通过注射口时间以及内外压差决定，变量因素单一，系统简单可控。
实施例一：基于本发明的微量单细胞建库测序
本实施例利用本发明从液滴内回收5000-10000细胞的单细胞转录组信息，证明方法和装置可以实现微量液滴和细胞的单细胞组学分析。具体步骤如下：
1、液滴制备
1)将NIH3T3(ATCC No.CCL-92)和293T细胞(ATCC No.CRL-3216)等量混合，取5000或10000混合细胞，使用专用溶液(50％RPMI1640培养基(Gibco)，30％0.4(w/v)Ficoll，18％胎牛血清，1％青霉素-链霉素溶液，1％F68表面活性剂)重悬至500或1000个每微升(μl)；
2)将DNBelab C4试剂盒RNA capture磁珠取出100μl，放置在磁力架(12321D，Invitrogen)，等待2分钟，丢弃上清；
3)取10μl细胞悬液重悬磁珠，加入DNBelab C4试剂盒CR试剂1μl，加入图3A所示装置102，上述专用溶液加入103，液滴生成油(1864005，Bio-rad)加入101，将30ml注射器(BD)拉杆从15毫升(ml)刻度处拉动至20ml，完成液滴制备。
4)液滴在室温静置2小时，模拟细胞反应所需时间。
2、注射试剂
1)取50μl DNBelab C4 Index Carrier放置在磁力架(12321D，Invitrogen)，等待2分钟，丢弃上清，加入细胞裂解液25μl重悬，使用100μl移液器调整至25μl移液体积，吸取IndexCarrier悬液，将装载悬液的吸头插入图3D的303孔；
2)使用100μl移液器吸取全部生成的液滴，静置5分钟，调整移液器量程，排出吸头下层的油，将吸头插入图3的302孔；
3)使用200μl移液器吸取液滴生成油，将吸头插入图3的301孔；
4)将图4所示芯片电极联通20kHz 200Vpp电压；
5)使用导管如图4所示连接，注射器拉杆由17ml拉动至20ml，开始液滴注射(图3D)；
6)20分钟后液滴注射完成；
7)液滴静置40分钟；
8)随后依照DNBelab C4 RNA建库步骤操作。
3、单细胞数据结果
表1 基于液滴注射的单细胞建库测序结果
Figure PCTCN2022081698-appb-000001
Figure PCTCN2022081698-appb-000002
#5000和10000细胞组各重复3次实验
与主流的单细胞建库测序方法相比(DNBelab C4常规操作步骤)，本方法和装置实现细胞在液滴内反应后的和主流单细胞建库测序方法对接，细胞回收率等参数与主流方法参数接近，表明本方法对现有方法的良好兼容性。
此外，本实施例与以往方法相比有着下列关键性改进：
表2 本实施例和其他技术对比
Figure PCTCN2022081698-appb-000003
实施例二：分选后微量液滴的单细胞建库测序
1、液滴制备
1)将NIH3T3和293T细胞等量混合，使用专用溶液(50％RPMI1640培养基(Gibco)，30％0.4(w/v)Ficoll，18％胎牛血清，1％青霉素-链霉素溶液，1％F68表面活性剂)重悬至1000个每微升(μl)；
2)将DNBelab C4试剂盒RNA capture磁珠取出400μl，放置在磁力架(12321D，Invitrogen)，等待2分钟，丢弃上清；
3)取90μl细胞悬液重悬磁珠，加入DNBelab C4试剂盒CR试剂10μl，加入图3A所示装置(WO2020063864A1，CN209144161U)加样孔102，103加入上述专用溶液，101加入液滴生成油(1864005，Bio-rad)，将注射器拉杆从15毫升(ml)刻度处拉动至20ml，完成液滴制备。
2、液滴分选
4)将液滴转移至1ml注射器，经导管连接至图3B 202；
5)按照图3B所示，通过导管将油连接至分选芯片201口，芯片出口203连接至液滴收集装置(图3C)；
6)将205电极连接至20kHz 1000Vpp正弦波电压，进行液滴分选；
7)待收集3000个液滴信号后，停止分选，将100μl移液器吸头及液滴转移至注射芯片；
8)注射试剂
9)取50μl DNBelab C4 Index Carrier放置在磁力架(12321D，Invitrogen)，等待2分钟，丢弃上清，加入细胞裂解液25μl重悬，使用100μl移液器调整至25μl移液体积，吸取IndexCarrier悬液，将装载悬液的吸头插入图3D的303孔；
10)将装有100μl液滴的吸头静置5分钟，调整移液器量程，排出吸头下层的油，将吸头插入图3D的302孔；
11)使用200μl移液器吸取液滴生成油，将吸头插入图3D的301孔；
12)将图4所示芯片电极联通20kHz 200Vpp电压；
13)注射器拉杆由17ml拉动至20ml，开始液滴注射；
14)20分钟后液滴注射完成；
15)液滴静置40分钟；
16)随后依照DNBelab C4 RNA建库步骤操作。
上述步骤6)中液滴分选的具体方法为：将液滴转移至1mL注射器，经导管连接入口202，同时将油相加入5mL注射器连接入口201。将盛有液滴和油相的注射器分别使用注射泵推动，液滴流经通道206。此处，阳性液滴会在激光激发下发光，液滴由此产生的光信号可以经电极产生偏转力，该偏转力会使液滴向203通道偏转；而阴性液滴经由204通道废弃。
3、建库cDNA文库浓度
本发明文库cDNA低于标准流程，但是显著高于分选液滴-破乳-标准化流程建库，解决微量液滴细胞无法回收的问题(表3)。
对比例1：
将3000细胞按照标准化DNBelab C4单细胞试剂盒标准建库流程进行建库，其他参数均与实施例2相同，结果如表3所示。
对比例2
将3000实施例1中制备的混合细胞依照破乳-细胞回收方法，其他参数均与实施例2相同，结果如表3所示，无法回收细胞进行单细胞建库，破乳-细胞回收方法具体包括如下步骤：
1)分别调整细胞悬液浓度至4×106/毫升和4×105/毫升；2)向图3A所示液滴生成装置的孔102加入50微升细胞悬液，孔103加入50微升工作液，孔101加入100微升液滴生成油，拉动注射器(从13毫升刻度处拉至20毫升刻度)，生成包含细胞的液滴，并通过导管，将生成的液滴导入液滴收集管；此时，由于细胞沉降，部分细胞沉积在PDMS微流控液滴生成装置加样口内，此部分细胞的丢失将影响后续统计细胞回收率。因此，当孔102内液面下降至孔高度约四分之一时，分别向孔102和103加入50微升DMEM培养基，使用移液器缓慢吹打孔102内液体3次，以重悬沉淀细胞；3)完成液滴生成后，上述液滴收集管内有两层，上层是油包水液滴，下层为少量液滴生成油；使用1ml注射器移除液滴收集管下层油；4)向液滴收集管内缓慢滴加200微升PFO，静置10分钟；5)此时样品收集管内有三层，下层是液滴生成油和PFO的油相混合物，中间是未破乳的液滴，上层是破乳释放的含细胞的水相，使用1ml注射器移除液滴收集管下层油相；6)向样品收集管内缓慢滴加100微升PFO，静置5分钟；7)将所述样品收集管在50g下离心10秒；8)此时样品收集管内液体分为两层，下 层是液滴生成油和PFO的油相混合物，上层是破乳释放的含细胞的水相，两层之间有清晰的界面，使用100微升量程移液器轻轻吹打上层细胞悬液，不触及界面；9)在液滴生成时，液滴内共包裹200微升液体；转移150微升上层细胞悬液至新1.5毫升EP管，剩余约50微升细胞悬液；10)向所述样品收集管中加入200微升DMEM培养基；11)轻轻吹打所述样品收集管的上层细胞悬液，转移200微升上层至1.5毫升EP管，此时有少量细胞残留，但是为了避免PFO污染细胞，影响后续实验(如RNA提取，PCR反应等)；12)对所述EP管中的细胞悬液进行细胞计数；13)取10微升上述细胞悬液与10微升AO/PI染液混合，并使用CountStar计数仪(CountStar Rigel S3)计算细胞活率。
表3 3000细胞基于本方法和标准化操作的单细胞建库结果
Figure PCTCN2022081698-appb-000004
#各重复3次实验
工业实用性
本装置的液滴注射芯片由单层PDMS胶制备键合，工艺成熟、操作简单、成本低廉；液体动力由注射器形成负压提供，液流稳定、设备成本低廉；使用时，可以使用移液器吸头改造为锥形管，可以实现最少10微升液滴加样，克服注射泵和芯片连接管内死体积造成的液滴损失，实现微量液滴无损或低损失加样。本芯片对流道高度和宽度存在要求，由于实际操作时可能使用硬质磁珠作为细胞遗传物质富集载体，需要流道宽度和高度分别达到磁珠直径2倍以上，但是为了避免液滴和注射口距离过远，导致无法注射，要求流道高度小于液滴直径，因此，芯片高度和宽度应该满足：高度/宽度>2*磁珠直径；同时，高度/宽度<液滴直径。注射相流速收到压强和液滴粘度影响，随着液体粘度增大，注入液滴的体积减少；本发明推荐使用0.4(w/v)Fiocll溶液调节注射相粘度，以调整液体注入体积；使用本发明所示芯片时，需要加入0.4(质量体积比，w/v)Ficoll溶液至Ficoll终浓度大于5％(w/v)，避免注射相浸润注射口附近PDMS。

Claims (13)

  1. 一种液滴收集装置，其特征在于，包括内管、外管和第一负压装置，所述内管的上端与待收集液滴相连通，下端与外管相连通；所述外管上端开口，所述内管套设在外管内；所述外管与第一负压装置相连通。
  2. 根据权利要求1所述的液滴收集装置，其特征在于，所述内管为上大下小的倒锥形管。
  3. 根据权利要求1所述的液滴收集装置，其特征在于，所述第一负压装置为注射器。
  4. 一种液滴注射装置，其特征在于，包括液滴注射芯片，所述液滴注射芯片上设有液滴生成油入口、液滴入口、注射试剂入口、液滴出口、主流道和微流道；所述液滴入口和液滴出口分别位于主流道的两端，液滴生成油入口和注射试剂入口通过微流道与主流道相连通；注射试剂入口与主流道的连通设有电极；所述液滴在主流道内依次接触液滴生成油和注射试剂；在液滴出口处形成包裹注射试剂的液滴。
  5. 根据权利要求4所述的液滴注射装置，其特征在于，所述液滴注射装置芯片的液滴入口与液滴收集装置相连通；液滴出口与液滴收集管相连通，所述液滴收集管与第二负压装置相连通。
  6. 根据权利要求4所述的液滴注射装置，其特征在于，所述第二负压装置为为注射器。
  7. 根据权利要求4所述的液滴注射装置，其特征在于，所述液滴中含有磁珠，所述主流道的宽度和高度中的一种大于2倍磁珠直径。
  8. 根据权利要求7所述的液滴注射装置，其特征在于，所述主流道的宽度和高度中的一种小于液滴直径。
  9. 一种微量液滴内单细胞测序文库构建装置，其特征在于，包括液滴生成芯片和权利要求4-8任一所述液滴注射装置，所述液滴生成芯片生成的液滴与与液滴注射装置的液滴入口相连通。
  10. 根据权利要求9所述的微量液滴内单细胞测序文库构建装置，其特征在于，还包括包括液滴分选芯片和权利要求1-3任一所述的液滴收集装置，所述所述液滴生成芯片生成的液滴经过液滴分选芯片与液滴收集装置的内管上端相连通，并经过液滴收集装置的收集后，与液滴注射装置的液滴入口相连通。
  11. 一种微量液滴内单细胞测序文库构建方法，其特征在于，使用权利要求9或10所述的微量液滴内单细胞测序文库构建装置。
  12. 根据权利要求11所述的微量液滴内单细胞测序文库构建方法，其特征在于，包括如下步骤：
    1)将待建库细胞、磁珠、液滴生成油加入液滴生成芯片，完成液滴的制备；
    2)将制备好的液滴连通至液滴注射装置的液滴入口，并分别在液滴生成油入口和注射试剂入口连通液滴生成油和注射试剂；
    3)将液滴注射装置的芯片电极接通电源，打开第二负压装置，收集融合了 注射试剂的液滴；
    4)将融合了注射试剂的液滴依照DNBelab C4 RNA建库步骤操作。
  13. 根据权利要求11所述的微量液滴内单细胞测序文库构建方法，其特征在于，在步骤1和步骤2之间包括分选收集步骤，具体如下：
    S1)将步骤1)生成的液滴的使用液滴分选芯片进行分选，得到分选后的混合液滴；
    S2)分选后的液滴经液滴收集装置的内管上入口进入内管，随着油和液滴流入内管，液滴悬浮在上层，油经内管底部流动至外管，当液滴上浮至内管口，拉动注射器抽取外管油令内管液面下降；
    S3)完成液滴收集后，拿出内管，将插入液滴注射装置的液滴入口，完成液滴注射装置的液滴入口与液滴的连通。
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CN104877899A (zh) * 2014-02-28 2015-09-02 中国科学院青岛生物能源与过程研究所 一种基于液滴的微生物快速直接绝对定量检测系统和方法
CN206052002U (zh) * 2016-08-31 2017-03-29 四川蓝光英诺生物科技股份有限公司 制备微球的装置
CN108080046A (zh) * 2018-01-29 2018-05-29 苏州昊通仪器科技有限公司 液滴生成装置和方法
CN112439467A (zh) * 2019-08-29 2021-03-05 北京怡天佳瑞科技有限公司 用于制备乳化液滴的芯片及装置
CN113026112A (zh) * 2019-12-25 2021-06-25 苏州绘真生物科技有限公司 用于构建人单细胞bcr测序文库的试剂盒及其应用
WO2022034344A1 (en) * 2020-08-14 2022-02-17 Cambridge Enterprise Limited Modular microfluidic methods and devices
CN112574937A (zh) * 2020-12-31 2021-03-30 江南大学 基于微流控芯片的超微注射法及其在高通量筛选中的应用

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