WO2023137583A1 - 确定多种物质与细胞作用关系的方法和微井阵列芯片 - Google Patents

确定多种物质与细胞作用关系的方法和微井阵列芯片 Download PDF

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WO2023137583A1
WO2023137583A1 PCT/CN2022/072517 CN2022072517W WO2023137583A1 WO 2023137583 A1 WO2023137583 A1 WO 2023137583A1 CN 2022072517 W CN2022072517 W CN 2022072517W WO 2023137583 A1 WO2023137583 A1 WO 2023137583A1
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droplet
microwell
array chip
micro
droplets
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PCT/CN2022/072517
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English (en)
French (fr)
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刘杨
谢润
王诗雨
高开
吕孟华
刘亚
刘龙奇
徐讯
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深圳华大生命科学研究院
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Priority to CN202280089454.3A priority Critical patent/CN118647871A/zh
Priority to PCT/CN2022/072517 priority patent/WO2023137583A1/zh
Publication of WO2023137583A1 publication Critical patent/WO2023137583A1/zh

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to the field of medicine. Specifically, the present invention proposes a method for determining the relationship between various substances and cells and a microwell array chip.
  • Precision medicine refers to tailoring the best treatment plan for patients according to the differences among different people, expecting to achieve the best treatment effect and the least side effects.
  • precise and personalized treatment has become one of the strategies to deal with cancer heterogeneity.
  • scientists have discovered many genes related to canceration in different tissues, such as Myc and Ras.
  • information on possible cancer-causing gene mutations can be obtained, so that treatment plans can be formulated accordingly.
  • personalized medicine can provide more effective and targeted treatment through more accurate diagnosis, prevent the occurrence of certain diseases, and save treatment costs more than current treatment methods.
  • in vitro response experiments of external substances can be performed on biopsy samples of patients to obtain the response effect of cancer cells to different substances, which can be used to assist in the formulation of personalized treatment plans.
  • external substances including compounds, hormones, antibodies and other substances that can act on cells
  • Microfluidic technology is a technology that can precisely control and manipulate micro-scale fluids. It has the ability to shrink experiments in the laboratory to a chip with a scale of several square centimeters. It is also an interdisciplinary subject including physics, engineering, chemistry and biology.
  • microfluidic chips are considered to be the most promising high-throughput single-cell analysis platform in the field of biology, especially in single-cell related research.
  • Analysis at the single-cell level has important research significance for early diagnosis and treatment of major diseases and drug screening, and has become a research hotspot in recent years.
  • the currently reported methods for drug screening at the single-cell level cannot achieve high-throughput drug screening. Therefore, a method that can study the effects of different drugs on single cells from the transcriptome level is needed to achieve large-scale drug screening in vitro, which is helpful for screening multiple drugs with synergistic effects, and provides assistance for personalized precision medicine and drug combination.
  • the present invention aims to solve the technical problems existing in the prior art at least to a certain extent. For this reason, the present invention proposes a method for determining the relationship between multiple substances and cells and a microwell array chip. Using this method, the relationship between multiple substances can be studied at the single-cell level, which is helpful for high-throughput screening of combined substances.
  • the present invention provides a method for determining the relationship between various substances and cells. According to an embodiment of the present invention, the method includes:
  • the first droplet is a mixed droplet containing a plurality of different molecularly coded droplets
  • the second droplet is a droplet containing a single cell
  • the third droplet is a droplet containing a single sequencing magnetic bead, cell lysate, and fragmentation reagent
  • each of the molecularly coded droplets contains a substance and its matching encoded nucleic acid molecule and index magnetic beads
  • there are multiple microwell combinations on the core of the microwell array each of which includes a large microwell and a plurality of large microwells Small micro-wells adjacent to and connected to the micro-wells, the aperture of the large micro-well is larger than the aperture of the small micro-well;
  • encoding nucleic acid molecules are used to encode and mark different substances, so as to facilitate subsequent analysis of sequencing results.
  • Both the material droplet and the magnetic bead droplet are small droplets, and the cell droplet is a large droplet.
  • the microwell array chip has connected microwells with different apertures, the microwells with small apertures can capture small droplets, and the microwells with large apertures can capture large droplets.
  • the second liquid droplet (also called “cell droplet”) with a larger pore size is first added to the chip and falls into the large and micro wells. Then add the first droplet with small pore size (also called “substance droplet”) to the chip, and each substance droplet will randomly fall into a small microwell, and then fuse one cell droplet with multiple substance droplets to complete the addition process, and take out the chip after culturing in the incubator for a certain period of time. Due to the effect of interfacial tension, the fused large droplets will occupy the large microwells, leaving the position of the small microwells free for loading of subsequent sequencing magnetic bead droplets.
  • the first droplet with small pore size also called “substance droplet”
  • the third droplet small droplet wrapped with sequencing magnetic beads, cell lysate and fragmentation reagent, also known as "magnetic bead droplet" is added to the small micro-well in the chip and fused with the large droplet to complete cell lysis and capture of mRNA, molecular coding and index sequences. Collect the fused droplets, break the emulsion and recover the magnetic beads, and carry out the subsequent single-cell library construction process with the nucleic acid information and index sequence information carried on the magnetic beads.
  • index magnetic beads carrying index sequences are added to each material droplet. Sequencing magnetic beads can capture the index sequence. Based on the type of the index sequence, it can be known whether it is derived from the same cell droplet, so that multiple substances that act on the same cell can be known, which helps to achieve high-throughput combination substance screening.
  • the above-mentioned method for determining the relationship between various substances and cells may also have the following additional technical features:
  • each molecular coding droplet contains 3-8 index magnetic beads, and each index magnetic bead contains a different index sequence.
  • the fragmentation reagent is selected from the group suitable for fragmentation of disulfide bonds.
  • the fragmentation reagent is selected from dithiothreitol, tris(2-carbonylethyl)phosphonium hydrochloride, tris(3-hydroxypropyl)phosphine and/or ⁇ -mercaptoethanol.
  • the second droplet and the third droplet are sorted by a sorting chip.
  • the fusion is electrical fusion or chemical fusion.
  • the diameter of the large and micro wells is 80-100 microns, and the depth is 60-80 microns; the diameter of the small and micro wells is 40-60 microns, and the depth is 60-80 microns.
  • the sequencing magnetic beads are suitable for capturing nucleic acid molecules and index sequences.
  • the microwell array chip provided in step (1) is subjected to surface plasma treatment, so that the grooves in the microwell array chip for the flow of droplets are bonded to the large microwells and small microwells, so as to facilitate the capture of droplets.
  • the method for collecting the second fused liquid droplets includes: turning the micro-well array chip by 180° so that the openings of the large micro-wells and the small micro-wells are upward, and adding oil to the micro-well array chip, so that the second fused liquid droplets flow out from the large micro-wells into a collection container.
  • the present invention provides a microwell array chip.
  • the microwell array chip includes: a microwell array layer, on which a plurality of microwell combinations are arranged, and each microwell combination includes a large microwell and a plurality of small microwells adjacent to and connected to the large microwell, the aperture of the large microwell is larger than the aperture of the small microwell; a channel layer, the channel layer is stacked with the microwell array layer, and grooves are arranged on the channel layer, and the openings of the large microwells and small microwells face the grooves.
  • the use of the microwell array chip according to the embodiment of the present invention can realize the study of the relationship between compound substances at the single-cell level, which is helpful to realize high-throughput compound substance screening, and is of great significance for the study of compound substances at the transcriptome level.
  • the diameter of the large and micro wells is 80-100 microns, and the depth is 60-80 microns; the diameter of the small and micro wells is 40-60 microns, and the depth is 60-80 microns.
  • the groove is connected to the large microwell or the small microwell through a chemical bond.
  • the microwell array chip is used to implement the aforementioned method for screening composite substances.
  • the method designed in the present invention uses molecular codes to encode substances under different conditions, and combines microfluidic technology to wrap single cells, nucleic acid capture magnetic beads, substances and corresponding codes in the same droplet.
  • single-cell sequencing technology it can realize high-throughput compound material screening at the single cell level, improve the efficiency of compound material screening, and reduce the consumption of manpower and material resources.
  • Fig. 1 shows a schematic flowchart of a method for determining the relationship between various substances and cells according to an embodiment of the present invention
  • Fig. 2 has shown the top view of microwell array chip structure according to one embodiment of the present invention
  • FIG. 3 shows a schematic diagram of a channel layer structure according to an embodiment of the present invention
  • Fig. 4 has shown the side view of microwell array chip structure according to one embodiment of the present invention.
  • Fig. 5 shows a schematic flow chart of droplet preparation according to one embodiment of the present invention
  • Figure 6 shows a schematic flow chart of droplet capture and fusion according to one embodiment of the present invention
  • Fig. 7 shows the physical picture (a) of the droplet capturing the large droplet and the physical picture (b) after capturing the small droplet according to one embodiment of the present invention, the scale is 100 microns;
  • Figure 8 shows a schematic diagram of the composition of sequencing magnetic beads capturing sequence information and substance coding according to an embodiment of the present invention
  • Figure 9 shows the fragment distribution diagram after specific amplification of the index sequence according to an embodiment of the present invention, and an obvious characteristic peak appears around 170bp;
  • Fig. 10 shows the fragment distribution diagram after specific amplification of the substance coding sequence according to an embodiment of the present invention, and an obvious characteristic peak appears at about 130 bp.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, a feature defined as “first” and “second” may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.
  • the present invention proposes a method for determining the relationship between various substances and cells and a microwell array chip, which will be described in detail below.
  • the present invention provides a method for determining the relationship between various substances and cells.
  • the present invention does not strictly limit the specific type of the term "substance”, which may be compounds, hormones, antibodies and other substances that can or cannot produce effects on the body.
  • the present invention generally embodies "substance” as "drug”.
  • the method for determining the relationship between various substances and cells includes:
  • S100 provides a first liquid droplet, a second liquid droplet, a third liquid droplet and a microwell array chip.
  • a first droplet, a second droplet, a third droplet, and a microwell array chip are provided, wherein the first droplet is a mixed droplet containing a plurality of different molecularly encoded droplets, the second droplet is a droplet containing a single cell, and the third droplet is a droplet containing a single sequencing magnetic bead (for capturing nucleic acid), a cell lysate, and a fragmentation reagent. well and a plurality of small and micro wells adjacent to and connected to the large and micro wells.
  • Fluorescent encoding technology refers to the use of the color of fluorescent dyes to encode solutions of different drugs and generate droplets, which are then mixed with droplets wrapped in single cells to achieve drug screening based on single cells.
  • this method is limited by the type of fluorescence and the detection device, which makes it difficult to screen a large number of drugs, and lacks information about gene expression inside the cell.
  • the molecular code contains fixed sequence UMI, specific sequence and magnetic bead capture sequence.
  • the preparation method of the first droplet includes: mixing a certain amount of molecular codes with corresponding substances and index magnetic beads, injecting them into a droplet generation chip to generate molecularly coded droplets of uniform size, and so on to generate droplets with different molecular codes. Finally, the generated droplets are collected and mixed in the same collection tube to complete the preparation of the material droplets.
  • Molecular codes are added for the purpose of distinguishing different substances. The reason for introducing indexed magnetic beads is mainly because multiple sequencing magnetic beads will be captured when the sequencing magnetic bead droplet is finally captured, so that the final fused droplet contains multiple sequencing magnetic beads.
  • index beads carrying index sequences are introduced, and under the action of fragmentation reagents, the index beads can be broken into index sequences. Since each index bead is different, this makes each final large droplet contain a variety of different index sequences. Through sequencing analysis, it can be determined which sequence beads capture the same type of index sequences, and it can be considered that these sequencing beads come from the same droplet. In this way, the mRNA information and molecular coding information captured by these sequencing beads are combined into one through an algorithm, so that a complete single-cell sequencing can be achieved. . Specifically, each molecular coding liquid droplet contains 3-8 index magnetic beads, and the index sequences contained on each index magnetic bead are different.
  • the preparation method of the second droplet includes: injecting a certain amount of cell suspension into the droplet generation chip, adjusting the cell concentration, so that the ratio of single cells wrapped in the droplet is high, and at the same time ensuring that the double-encapsulation rate is at a low level, and then sorting the generated droplet by using dielectrophoresis and other methods through the sorting chip to obtain a droplet containing a single cell.
  • the preparation method of the third liquid droplet includes: injecting a certain amount of sequencing magnetic bead suspension (obtained by resuspending the sequencing magnetic beads with a cell lysate and a fragmentation reagent) into the droplet generating chip, adjusting the concentration of the sequencing magnetic beads so that the ratio of a single sequencing magnetic bead wrapped in the droplet is high, while ensuring that the double packet rate is at a low level, and then sorting the generated liquid droplets by dielectrophoresis and other methods through the sorting chip to obtain a droplet containing a single sequencing magnetic bead.
  • the "big” and “small” descriptions involved in the "big micro well” and “small micro well” described in the present invention refer to the pore size of the micro well, and the pore size of the large micro well is larger than that of the small micro well.
  • the diameter of the large and micro wells is 80-100 microns, and the depth is 60-80 microns; the diameter of the small and micro wells is 40-60 microns, and the depth is 60-80 microns.
  • large microwells can capture single-cell droplets, and small microwells can capture material droplets and magnetic bead droplets.
  • the fragmentation reagent is selected from a group suitable for fragmentation of disulfide bonds.
  • the index sequence is connected to the magnetic bead through a disulfide bond, and the disulfide bond is broken by a breaking reagent, so that the index sequence carried on the index magnetic bead is broken from the magnetic bead. Since the sequencing magnetic bead contains a sequence that matches the index sequence, the broken index sequence can be captured, which is convenient for subsequent sequencing and classification, and multiple substances that act on the same cell can be known.
  • the fragmentation reagent is selected from dithiothreitol (DTT), tris(2-carbonylethyl)phosphonium hydrochloride (TCEP), tris(3-hydroxypropyl)phosphine (THPP) and/or ⁇ -mercaptoethanol.
  • DTT dithiothreitol
  • TCEP tris(2-carbonylethyl)phosphonium hydrochloride
  • THPP tris(3-hydroxypropyl)phosphine
  • ⁇ -mercaptoethanol ⁇ -mercaptoethanol
  • S200 firstly adds the second liquid drop into the chip and falls into the large micro wells, and then adds the first liquid drop into the chip and falls into multiple small micro wells.
  • the second droplet is added into the microwell array chip and falls into the large microwells, and then the first droplet is added into the microwell array chip and falls into multiple small microwells.
  • the microwell array chip provided in step S100 is subjected to surface plasma treatment, so that the grooves in the microwell array chip for the flow of liquid droplets are bonded to the large microwells and small microwells, so as to facilitate the capture of liquid droplets.
  • bonding used in the present invention refers to the technology of directly bonding two pieces of homogeneous or heterogeneous semiconductor materials with clean and atomically flat surfaces under certain conditions after surface cleaning and activation treatment, and bonding the wafers into one body through van der Waals force, molecular force or even atomic force.
  • the surface of the cured chip (also known as "PDMS substrate") has a certain degree of adhesion, and a pair of formed PDMS substrates can be naturally bonded with the help of intermolecular attraction without any treatment. However, this bonding strength is limited and liquid leakage is prone to occur.
  • the surface of PDMS after plasma treatment introduces hydrophilic -OH groups and replaces -CH groups, so that the surface of PDMS exhibits extremely strong hydrophilic properties. After the two layers of PDMS are bonded together, the following reaction occurs between the Si-OH on the two surfaces: A strong Si-O bond was formed between the two layers of PDMS, thus completing the irreversible bonding between the two layers.
  • the first droplet and the second droplet are fused to obtain the first fused droplet.
  • the first fused droplet occupies the large microwell, and the small microwell is vacated, and the microwell array chip is used for cell culture.
  • the first fused droplets after fusion mainly occupy the large microwells, leaving the position of the small microwells free for the loading of subsequent sequencing magnetic bead droplets.
  • the present invention does not strictly limit the fusion method of the two droplets.
  • it can be realized by using an electric field to destroy the stability of the interface, or by using chemical reagents such as perfluorobutanol, which can be flexibly selected according to actual needs.
  • the third droplet after the cell culture is completed, the third droplet is added to the vacated microwell, the third droplet will fuse with the first fused droplet after cell culture, and cell rupture will occur under the action of the cell lysate in the third droplet, and the nucleic acid molecule in the cell and the encoded nucleic acid molecule will be captured by the sequencing magnetic beads.
  • the small droplets wrapped with sequencing magnetic beads, cell lysate and fragmentation reagent are added to the chip and fused with the first fusion droplet to obtain the second fusion droplet, thereby completing cell lysis, capture of mRNA, molecular coding and index sequences.
  • the second fusion droplet is demulsified, the sequencing magnetic beads are collected, and the nucleic acid and index sequence carried on the sequencing magnetic beads are constructed and sequenced, based on determining the relationship between various substances and cells.
  • the method for collecting the second fused liquid droplet includes: turning the microwell array chip by 180°, so that the openings of the large microwell and the small microwell are upward, and adding oil to the microwell array chip, so that the second fused liquid droplet flows out from the large microwell into the collection container.
  • the microwell array chip includes a stacked microwell array layer and a channel layer, large microwells and small microwells are arranged on the microwell array layer, and the openings of the microwells face the grooves provided on the channel layer for liquid flow.
  • the chip Since the density of the water phase is lower than that of the oil phase, the second fused liquid droplet floats above the groove, and the second fused liquid droplet cannot be taken away by adding oil to the groove. Therefore, the chip needs to be turned 180° so that the openings of the large microwell and the small microwell are upward, so that the second fusion droplet will float in the groove, and oil can be used to push out the droplet separated from the microwell and collect it in a centrifuge tube.
  • the magnetic beads are recovered by breaking the emulsion to carry out the subsequent single-cell library construction process, and the corresponding relationship between single cells and drugs is obtained by splitting the sequencing information, thereby realizing high-throughput screening of drugs at the single-cell level.
  • the present invention provides a microwell array chip.
  • the microwell array chip includes: a microwell array layer 100 and a channel layer 200 .
  • the micro-well array layer 100 is provided with a plurality of micro-well combinations, each micro-well combination includes a large micro-well 110 and a plurality of small micro-wells 120 adjacent to and connected to the large micro-well 110, and the aperture of the large micro-well 110 is greater than the aperture of the small micro-well 120.
  • Both the drug droplet and the magnetic bead droplet are small droplets, and the cell droplet is a large droplet.
  • the microwell array layer has connected microwells with different apertures, the microwells with small apertures can capture small droplets, and the microwells with large apertures can capture large droplets.
  • the diameter of the large and micro wells is 80-100 microns, and the depth is 60-80 microns; the diameter of the small and micro wells is 40-60 microns, and the depth is 60-80 microns.
  • large microwells can capture single-cell droplets, and small microwells can capture drug droplets and magnetic bead droplets.
  • the channel layer 200 is stacked with the microwell array layer 100, the channel layer is provided with grooves 210, and the openings of the large microwells 110 and the small microwells 120 face the grooves 210. Since the micro-wells and grooves of the micro-well array layer have been pre-treated with surface plasma, the two can be bonded into one body, thereby avoiding the leakage of the added liquid. Adding liquid droplets to the grooves, since the liquid droplets are lighter than oil, under the action of buoyancy, the microwells can capture liquid droplets.
  • the groove 210 is connected with the large microwell 110 or the small microwell 120 through a chemical bond.
  • liquid droplets that flow into the grooves can be captured by the microwells.
  • the microwell array chip is used to implement the aforementioned method for screening drugs.
  • the features and advantages described above for the method for screening drugs are also applicable to the microwell array chip, and will not be repeated here.
  • Step 1 This embodiment adopts a droplet generating device similar to CN209144161U, wherein the droplet generation chip is replaced with the chip involved in patent WO2020063864A1, and the syringe is replaced with a BD 30ml syringe.
  • the schematic diagram of droplet generation is shown in Figure 5. Refer to the following steps to generate cell droplets, drug droplets and sequencing magnetic bead droplets.
  • a certain amount of molecular codes, corresponding drugs and index magnetic beads carrying index sequences are mixed and injected into the droplet generation chip to generate molecularly coded droplets of uniform size, and so on to generate droplets with different molecular codes. Finally, the generated droplets are collected and mixed in the same collection tube to complete the preparation of the drug droplets.
  • a certain amount of cell suspension is injected into the droplet generation chip, and by adjusting the cell concentration, the ratio of single cells wrapped in the droplet is high while ensuring that the double-wrapped rate is at a low level, and then the generated droplets are sorted through the cell sorting chip using dielectrophoresis and other methods to obtain droplets containing single cells.
  • a certain amount of sequencing magnetic bead suspension (obtained by resuspending sequencing magnetic beads in cell lysate) is injected into the droplet generation chip.
  • concentration of sequencing magnetic beads By adjusting the concentration of sequencing magnetic beads, the ratio of encapsulating a single sequencing magnetic bead in the droplet is high while ensuring a low double-package rate.
  • the generated droplets are sorted by dielectrophoresis and other methods through the sorting chip to obtain droplets containing a single sequencing magnetic bead.
  • the microwell chip as shown in Figures 1 to 4 is used to capture droplets.
  • the microwell chip includes a microwell array layer and a channel layer.
  • the microwell array layer has 28,800 connected microwells (apertures of 90 microns and 50 microns respectively), and the depth of the microwells is 70 microns. This ensures that a large microwell can only capture a large droplet, and a small microwell can only capture a small droplet, realizing the 1:1 pairing of droplets. Since the density of the water phase is lower than that of the oil phase, the liquid droplets are floating on the upper layer of the oil phase at this time.
  • the chip In order to capture the liquid droplets, the chip needs to be turned 180° in actual use, so that the micro-well head is flushed down, and it is bonded to the channel layer shown in Figure 2 through surface plasma treatment. At this time, the channel contains more than 25,000 effective micro-wells.
  • small droplets 50 microns in diameter
  • small droplets 50 microns in diameter
  • molecular codes molecular codes
  • indexing magnetic beads are loaded into the microwell array chip, so that different types of small droplets are randomly captured by the small microwells, so that each group of microwells will randomly capture two small material droplets and one large single-cell droplet.
  • oil is injected to push away the excess droplets. As shown in Figure 6c and Figure 7b, the droplet capture efficiency is above 95%.
  • the chip Place the chip on a shaker and shake slightly to allow the same group of droplets to collide.
  • the fused droplets randomly contain one or two substances and the corresponding molecular codes, as well as index magnetic beads (the index sequence is not broken, because no fragmentation reagent is added).
  • the fusion makes the positions of two small droplets vacant, and the fusion efficiency is above 80%.
  • the sequencing magnetic bead droplets contain cell lysate and index sequence fragmentation reagent, the cells will be lysed, and at the same time, the fragmentation reagent will fragment the index sequence on the index magnetic beads. In this way, the sequencing magnetic beads will capture the mRNA produced by cell lysis, the index sequence broken by the index magnetic beads, and the molecular code corresponding to the substance.
  • Step 3 After the droplet incubation is completed, the microfluidic chip is taken out, and then the sequencing magnetic bead droplet is injected. At this time, the droplet will be captured by the vacated small microwell again, and then the excess droplet is pushed out with oil. The state of the droplet at this time is shown in Figure 6 e. Finally, the process of droplet fusion in step 2 is repeated, and the state of the droplet at this time is shown in f of FIG. 6 .
  • Step 4 In order to recover the secondary fused droplets, the microwell array chip needs to be turned over 180°, and then the droplets detached from the microwells are pushed out of the chip with oil, and the droplets are collected in a centrifuge tube. As shown in Figure 8, since the sequencing magnetic bead droplets contain cell lysate, once the droplets are fused for the second time, the cells will be lysed to release the mRNA in the cells. At this time, the sequencing magnetic beads will capture all nucleic acid information including mRNA, molecular coding and index sequences. Finally, the BGI single-cell sequencing process was carried out, and the magnetic beads labeled with the same type of index sequence were classified as originating from the same droplet through bioinformatics analysis.

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Abstract

确定多种物质与细胞作用关系的方法,包括:提供第一液滴、第二液滴、第三液滴和微井阵列芯片,微井阵列芯片上有多个微井组合,每个微井组合包括一个大微井(110)和多个与大微井(110)相邻且连通的小微井(120),大微井(110)的孔径大于小微井(120)的孔径(S100);先将第二液滴加入微井阵列芯片内并落入大微井(110)内,再将第一液滴加入微井阵列芯片内并落入多个小微井(120)内(S200);使第一液滴和第二液滴发生融合,将微井阵列芯片进行细胞培养(S300);细胞培养完成后,将第三液滴加入空出的小微井(120)内,第三液滴将与细胞培养后的第一融合液滴发生融合,得到第二融合液滴(S400);将第二融合液滴进行破乳,收集磁珠,将磁珠上携带的核酸进行建库和测序,基于测序结果确定多种物质与细胞作用关系(S500)。

Description

确定多种物质与细胞作用关系的方法和微井阵列芯片 技术领域
本发明涉及医药领域。具体地,本发明提出了确定多种物质与细胞作用关系的方法和微井阵列芯片。
背景技术
精准医疗是指根据不同人之间的差异为病人量身设计出最佳的治疗方案,期望达到最佳的治疗效果和最小的副作用。而随着癌症的逐年肆虐,使得精准个性化的治疗成为应对癌症异质性的策略之一。借助于近十年来测序技术的迅速发展,科学家们已经发现了许多不同组织的癌变有关的基因,如Myc、Ras等。通过对患者的活检样本进行基因测序,可以获得可能的致癌的基因突变信息,从而相应地制定治疗方案。从长远角度看,个性化医疗通过更精确的诊断,提供更有效、更有针对性的治疗,预防某种疾病的发生,比目前的治疗手段更节约治疗成本。然而,对于测序结果和癌细胞对各种外加物质(包括化合物、激素、抗体等能对细胞产生作用的物质)的响应之间的关系,了解依然非常有限,因此测序结果对个性化治疗方案制定起到的指导作用,目前也较为有限。
为了解决这一问题,可以通过对患者的活检样本进行体外的外加物质(包括化合物、激素、抗体等能对细胞产生作用的物质)响应实验,获得癌细胞对不同物质的反应效果,用以辅助个性化治疗方案的制定。为了治疗的有效性,需要对大量的疑似有效的物质进行筛选,高通量的要求在多种药物筛选中更为突出。
近年来,DNA编码技术及测序技术不断发展,人们将DNA条形码引入单细胞测序上,从而实现了在单细胞水平上的高通量测序。同时这些高通量单细胞测序的实现也离不开微流控技术的发展。微流控技术是一种能够精确控制和操控微尺度流体的技术,具有能将在实验室中进行的实验微缩到一个尺度在几平方厘米的芯片上的能力,其同时还是一个包括了物理学、工程学、化学和生物学等在内的交叉学科。由于微流控芯片的微结构与单细胞尺寸相当,在生物学领域尤其是单细胞相关的研究中,微流控芯片被认为是最具有发展潜力的高通量单细胞分析平台。在单细胞水平上分析对重大疾病的早期诊断治疗以及药物筛选等方面具有重要的研究意义,已成为近年来研究的热点。目前报道的在单细胞水平上进行药物筛选的方法无法实现高通量的药物筛选,因此需要一种能从转录组水平研究不同药物对单细胞作用的方法,实现大规模的药物体外筛选,有助于筛选出协同作用的多种药物,为个性化精准医疗、联合用药提供帮助。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题。为此,本发明提出了一种确定多种物质与细胞作用关系的方法和微井阵列芯片,利用该方法可以在单细胞水平上研究多种物质的作用关系,有助于实现高通量组合物质筛选,对于从转录组水平上的组合物质研究和指导联合用药具有重要意义。
在本发明的一个方面,本发明提出了一种确定多种物质与细胞作用关系的方法。根据本发明的实施例,所述方法包括:
(1)提供第一液滴、第二液滴、第三液滴和微井阵列芯片;其中,所述第一液滴为含有多个不同分子编码液滴的混合液滴,所述第二液滴为含有单个细胞的液滴,所述第三液滴为含有单个测序磁珠、细胞裂解液和断裂试剂的液滴,每个所述分子编码液滴中含有一种物质及与其匹配的编码核酸分子和索引磁珠,所述微井阵列芯上有多个微井组合,每个所述微井组合包括一个大微井和多个与所述大微井相邻且连通的小微井,所述大微井的孔径大于小微井的孔径;
(2)先将所述第二液滴加入所述微井阵列芯片内并落入大微井内,再将所述第一液滴加入所述微井阵列芯片内并落入多个小微井内;
(3)使所述第一液滴和第二液滴发生融合,得到第一融合液滴,所述第一融合液滴占据在所述大微井内,所述小微井空出,将所述微井阵列芯片进行细胞培养;
(4)所述细胞培养完成后,将所述第三液滴加入所述空出的小微井内,将所述第三液滴与所述细胞培养后的第一融合液滴发生融合,在所述第三液滴中的细胞裂解液作用下发生细胞破裂,细胞内的核酸分子与所述编码核酸分子一同被测序磁珠所捕获,同时,在所述第三液滴中的断裂试剂作用下所述索引磁珠上的索引序列将断裂下来,也被测序磁珠所捕获,得到第二融合液滴,收集所述第二融合液滴;
(5)将所述第二融合液滴进行破乳,收集测序磁珠,将所述测序磁珠上携带的核酸和索引序列进行建库和测序,基于测序结果确定多种物质与细胞作用关系。
根据本发明实施例的方法中,利用编码核酸分子(亦称为“分子编码”)将不同的物质进行编码标记,以便于后续的测序结果分析。物质液滴与磁珠液滴都是小液滴,细胞液滴是大液滴。微井阵列芯片具有不同孔径的连通微井,孔径小的微井可以捕获小的液滴,孔径大的微井可以捕获大的液滴。
首先将孔径较大的第二液滴(亦称为“细胞液滴”)先加入芯片内并落入大微井内。随后将孔径小的第一液滴(亦称为“物质液滴”)加入芯片,每个物质液滴会随机落入一个小 微井中,接着将1个细胞液滴和多个物质液滴融合,完成添加过程,将芯片在培养箱中培养一定时间后取出。由于界面张力的作用,融合后的大液滴将占据大微井,而将小微井的位置空出,用于后续测序磁珠液滴的加载。物质处理完成后,将第三液滴(包裹了测序磁珠、细胞裂解液和断裂试剂的小液滴,亦称为“磁珠液滴”)加入芯片内的小微井中,并使其与大液滴融合,从而完成细胞的裂解及mRNA、分子编码和索引序列的捕获。收集融合后的液滴,破乳回收磁珠,将磁珠上携带的核酸信息和索引序列信息进行后续的单细胞建库流程。
由于有多个物质液滴共同作用于一个细胞液滴,并有多个测序磁珠(数量与物质液滴数量一致)捕获细胞培养后的核酸信息,为了获知不同测序磁珠是否作用于同一个细胞液滴,则在每一个物质液滴中加入携带有索引序列的索引磁珠。测序磁珠可以捕获索引序列,基于索引序列的类型可以获知是否来源于同一细胞液滴,从而可以获知作用于同一细胞的多个物质,有助于实现高通量组合物质筛选,对于从转录组水平上的组合物质研究和指导联合用药具有重要意义。
根据本发明的实施例,上述确定多种物质与细胞作用关系的方法还可以具有下列附加技术特征:
根据本发明的实施例,每个所述分子编码液滴中含有3~8个索引磁珠,每个所述索引磁珠上含有的索引序列不同。
根据本发明的实施例,所述断裂试剂选自适于断裂二硫键。
根据本发明的实施例,所述断裂试剂选自二硫苏糖醇、三(2-羰基乙基)磷盐酸盐、三(3-羟基丙基)膦和/或β-硫基乙醇。
根据本发明的实施例,所述第二液滴和第三液滴通过分选芯片进行分选得到。
根据本发明的实施例,所述融合为电融合或者化学融合。
根据本发明的实施例,所述大微井的孔径为80~100微米,深度为60~80微米;所述小微井的孔径为40~60微米,深度为60~80微米。
根据本发明的实施例,所述测序磁珠适于捕获核酸分子和索引序列。
根据本发明的实施例,进行步骤(2)之前,将步骤(1)所提供的微井阵列芯片进行表面等离子处理,以使所述微井阵列芯片中供液滴流动的凹槽与所述大微井和小微井发生键合,以便于捕获液滴。
根据本发明的实施例,所述收集第二融合液滴的方法包括:将所述微井阵列芯片翻转180°,以使大微井和小微井的开口向上,向所述微井阵列芯片中加入油,以使所述第二融合液滴从所述大微井内流出至收集容器中。
在本发明的又一方面,本发明提出了一种微井阵列芯片。根据本发明的实施例,所述微井阵列芯片包括:微井阵列层,所述微井阵列层上设置有多个微井组合,每个所述微井组合包括一个大微井和多个与所述大微井相邻且连通的小微井,所述大微井的孔径大于小微井的孔径;通道层,所述通道层与所述微井阵列层层叠设置,所述通道层上设置有凹槽,所述大微井和小微井的开口朝向所述凹槽。如前所述,利用根据本发明实施例的微井阵列芯片可以实现在单细胞水平上研究组合物质的作用关系,有助于实现高通量组合物质筛选,对于从转录组水平上的组合物质研究具有重要意义。
根据本发明的实施例,所述大微井的孔径为80~100微米,深度为60~80微米;所述小微井的孔径为40~60微米,深度为60~80微米。
根据本发明的实施例,所述凹槽与所述大微井或小微井通过化学键相连。
根据本发明的实施例,所述微井阵列芯片用于实施前面所述筛选组合物质的方法。
本发明所设计的方法利用分子编码将不同条件下的物质进行编码,结合微流控技术将单个细胞、核酸捕获磁珠与物质及对应的编码包裹在同一个液滴中,通过单细胞测序技术,能够实现单一细胞水平的高通量组合物质筛选,提高了组合物质筛选效率,减少人力和物力的消耗。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1显示了根据本发明一个实施例的确定多种物质与细胞作用关系的方法流程示意图;
图2显示了根据本发明一个实施例的微井阵列芯片结构的俯视图;
图3显示了根据本发明一个实施例的通道层结构示意图;
图4显示了根据本发明一个实施例的微井阵列芯片结构的侧视图;
图5显示了根据本发明一个实施例的液滴制备流程示意图;
图6显示了根据本发明一个实施例的液滴捕获和融合的流程示意图;
图7显示了根据本发明一个实施例的液滴捕获大液滴的实物图(a)及捕获小液滴后的实物图(b),标尺为100微米;
图8显示了根据本发明一个实施例的测序磁珠捕获序列信息以及物质编码的构成示意图;
图9显示了根据本发明一个实施例的索引序列特异性扩增后的片段分布图,在170bp处左右出现明显的特征峰;
图10显示了根据本发明一个实施例的物质编码序列特异性扩增后的片段分布图,在130bp左右处出现明显的特征峰。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
需要说明的是,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地,在本发明的描述中,除非另有说明,“多个”的含义是两个或两个以上。
本发明提出了一种确定多种物质与细胞作用关系的方法和微井阵列芯片,下面将分别对其进行详细描述。
确定多种物质与细胞作用关系的方法
在本发明的一个方面,本发明提出了一种确定多种物质与细胞作用关系的方法。本发明对于术语“物质”的具体类型不做严格限定,可以为化合物、激素、抗体等能对机体产生或不能产生作用的物质。为了方便理解和描述,本发明通常将“物质”可以具体化为“药物”。
根据本发明的实施例,参见图1,该确定多种物质与细胞作用关系的方法包括:
S100提供第一液滴、第二液滴、第三液滴和微井阵列芯片。
在该实施例中,提供第一液滴、第二液滴、第三液滴和微井阵列芯片,其中,第一液滴为含有多个不同分子编码液滴的混合液滴,第二液滴为含有单个细胞的液滴,第三液滴为含有单个测序磁珠(用于捕获核酸)、细胞裂解液和断裂试剂的液滴,每个分子编码液滴中含有一种物质及与其匹配的编码核酸分子(亦称为“分子编码”),微井阵列芯上有多个微井组合,每个微井组合包括一个大微井和多个与大微井相邻且连通的小微井。
荧光编码技术是指利用荧光染料的颜色,对不同药物的溶液进行编码并生成液滴,然后与包裹了单细胞的液滴混合,实现了基于单细胞的药物筛选。但是,该方法受限于荧光种类及检测装置,使得该技术很难实现大量药物的筛选,并缺少细胞内部基因表达的相关信息。
本申请中,采用分子编码设计,基于国际专利WO2021147069A1中引入的外源序列信 息,共有M×N种编码(M=4 10,N=4 10)。通过合成该序列作为药物编码,一种条件下的药物对应一种编码。例如,分子编码中含有固定序列UMI、特异性序列和磁珠捕获序列。
根据本发明的实施例,第一液滴的制备方法包括:将一定量的分子编码与对应的物质和索引磁珠混合后注入液滴生成芯片,生成尺寸均一的分子编码液滴,以此类推生成不同分子编码的液滴。最后将生成的液滴收集混合在同一个收集管中即完成物质液滴的制备。分子编码的添加目的是区分不同物质。之所以要引入索引磁珠,主要是因为最后捕获测序磁珠液滴的时候会捕获住多个测序磁珠,这样使得最终融合后的液滴中含有多个测序磁珠,若是不加索引序列,会导致最终测序无法判断哪两个测序磁珠来自于同一个液滴,会使测序磁珠捕获的mRNA信息无法归到一起去,即,无法判断哪几种药物是作用于同一细胞的,这就使得单细胞测序数据不完整无法使用。为了解决这一问题,引入了携带有索引序列的索引磁珠,在断裂试剂的作用下可使索引磁珠断裂出索引序列,由于每个索引磁珠都不一样,这使得每个最终的大液滴中含有多种不同的索引序列,通过测序分析判断哪几个测序磁珠上捕获的索引序列的种类一致,就可以认为这几个测序磁珠来源于同一个液滴,这样再通过算法将这几个测序磁珠的捕获的mRNA信息和分子编码信息合并成一个,这样就可以实现一个完整单细胞的测序。具体地,每个分子编码液滴中含有3~8个索引磁珠,每个索引磁珠上含有的索引序列不同。
第二液滴的制备方法包括:将一定量的细胞悬液注入到液滴生成芯片,通过调节细胞浓度,使得液滴中包裹单个细胞的比率较高,同时保证双包率处于较低的水平,再通过分选芯片利用介电泳等方法将生成的液滴进行分选,得到含有单个细胞的液滴。
第三液滴的制备方法包括:将一定量的测序磁珠悬液(利用细胞裂解液和断裂试剂重悬测序磁珠得到的)注入到液滴生成芯片,通过调节测序磁珠浓度,使得液滴中包裹单个测序磁珠的比率较高,同时保证双包率处于较低的水平,再通过分选芯片利用介电泳等方法将生成的液滴进行分选,得到含有单个测序磁珠的液滴。
需要说明的是,本发明所描述的“大微井”和“小微井”中涉及的“大”和“小”描述是指微井的孔径大小,大微井的孔径大于小微井的孔径。根据本发明的实施例,大微井的孔径为80~100微米,深度为60~80微米;小微井的孔径为40~60微米,深度为60~80微米。由此,大微井可捕获单细胞液滴,小微井可捕获物质液滴和磁珠液滴。
根据本发明的实施例,断裂试剂选自适于断裂二硫键。索引序列通过二硫键连接在磁珠上,采用断裂试剂断裂二硫键,从而使索引磁珠上携带的索引序列从磁珠上断裂下来,由于测序磁珠上含有与索引序列相匹配的序列,所以可以捕获断裂下来的索引序列,便于后续的测序和分类,获知作用于同一细胞的多个物质。根据本发明的具体实施例,断裂试 剂选自二硫苏糖醇(DTT)、三(2-羰基乙基)磷盐酸盐(TCEP)、三(3-羟基丙基)膦(THPP)和/或β-硫基乙醇。由此,可以特异性地断裂二硫键,且不影响核酸分子结构。
S200先将第二液滴加入芯片内并落入大微井内,再将第一液滴加入芯片内并落入多个小微井内。
在该实施例中,先将第二液滴加入微井阵列芯片内并落入大微井内,再将第一液滴加入微井阵列芯片内并落入多个小微井内。
根据本发明的实施例,进行步骤S200之前,将步骤S100所提供的微井阵列芯片进行表面等离子处理,以使微井阵列芯片中供液滴流动的凹槽与大微井和小微井发生键合,以便于捕获液滴。
本发明所使用的术语“键合”是指将两片表面清洁、原子级平整的同质或异质半导体材料经表面清洗和活化处理,在一定条件下直接结合,通过范德华力、分子力甚至原子力使晶片键合成为一体的技术。
固化后的芯片(亦称为“PDMS基片”)表面具有一定的粘附力,一对成型后的PDMS基片不加任何处理,即可借助分子间的引力自然粘合,但这种粘合强度有限,容易发生漏液。等离子体处理后的PDMS,其表面引入了亲水性质的-OH基团,并代替了-CH基团,从而使PDMS表面表现出极强的亲水性质。将处理后的两层PDMS相贴合,两表面的Si-OH之间发生如下反应:
Figure PCTCN2022072517-appb-000001
在两层PDMS之间形成了牢固的Si-O键结合,从而完成了二者间不可逆键合。
S300第一液滴和第二液滴融合并占据大微井,小微井空出,细胞培养。
在该实施例中,使第一液滴和第二液滴发生融合,得到第一融合液滴,第一融合液滴占据在大微井内,小微井空出,将微井阵列芯片进行细胞培养。
由于界面张力的作用,融合后的第一融合液滴主要占据大微井,而将小微井的位置空出,用于后续测序磁珠液滴的加载。
需要说明的是,本发明对于两种液滴的融合方式不做严格限定,例如可以使用电场破坏界面的稳定性来实现,亦可使用化学试剂如全氟丁醇等完成,具体可以根据实际需要灵活选择。
S400将第三液滴加入小微井内,与第一融合液滴发生融合,收集得到的第二融合液滴。
在该实施例中,细胞培养完成后,将第三液滴加入空出的小微井内,第三液滴将与细胞培养后的第一融合液滴发生融合,在第三液滴中的细胞裂解液作用下发生细胞破裂,细胞内的核酸分子与编码核酸分子一同被测序磁珠所捕获,同时,在第三液滴中的断裂试剂作用下索引磁珠上的索引序列将断裂下来,也被测序磁珠所捕获,得到第二融合液滴,收 集第二融合液滴。
药物处理完成后,将包裹了测序磁珠、细胞裂解液和断裂试剂的小液滴加入芯片内,并使其与第一融合液滴融合,得到第二融合液滴,从而完成细胞的裂解、mRNA、分子编码和索引序列的捕获。
S500破乳、建库、测序
在该实施例中,将第二融合液滴进行破乳,收集测序磁珠,将测序磁珠上携带的核酸和索引序列进行建库和测序,基于确定多种物质与细胞作用关系。
根据本发明的实施例,收集第二融合液滴的方法包括:将微井阵列芯片翻转180°,以使大微井和小微井的开口向上,向微井阵列芯片中加入油,以使第二融合液滴从大微井内流出至收集容器中。
微井阵列芯片包括层叠设置的微井阵列层和通道层,大微井和小微井设置于微井阵列层上,且微井开口朝向通道层上设置的供液体流动的凹槽。
由于水相的密度低于油相,因此第二融合液滴飘在凹槽上方,无法通过向凹槽内加油以将第二融合液滴带走。因此,芯片需要翻转180°,使得大微井和小微井的开口向上,这样第二融合液滴将飘在凹槽内,用油可将从微井中脱离的液滴推出,收集于离心管中。再破乳回收磁珠进行后续的单细胞建库流程,通过测序信息拆分得到单细胞和药物的对应关系,从而实现了单细胞水平上的药物高通量筛选。
本发明的筛选药物的方法还可以具有下列优点:
1)高通量:通过额外引入分子编码,可以实现多种药物条件(包括药物种类、药物浓度等)的编码;通过额外引入索引磁珠,可以区分作用于同一细胞的多种物质,以便于研究多种物质对细胞的作用效果,有助于联合用药;
2)省时省力:通过利用液滴微流控技术不但可以减少试剂用量,还可以避免繁琐的手工操作,提高筛选效率;
3)精确度高:由于本发明针对的是单细胞分析,通过引入分子编码,结合单细胞测序手段可以得到单个细胞转录组和药物的对应关系,从而更加精确地展示出不同条件下药物对单细胞转录组的影响,更具有研究价值。
微井阵列芯片
在本发明的另一方面,本发明提出了一种微井阵列芯片。根据本发明的实施例,参见图2,该微井阵列芯片包括:微井阵列层100和通道层200。
微井阵列层100上设置有多个微井组合,每个微井组合包括一个大微井110和多个与 大微井110相邻且连通的小微井120,大微井110的孔径大于小微井120的孔径。药物液滴与磁珠液滴都是小液滴,细胞液滴是大液滴。微井阵列层具有不同孔径的连通的微井,孔径小的微井可以捕获小的液滴,孔径大的微井可以捕获大的液滴。
根据本发明的实施例,大微井的孔径为80~100微米,深度为60~80微米;小微井的孔径为40~60微米,深度为60~80微米。由此,大微井可捕获单细胞液滴,小微井可捕获药物液滴和磁珠液滴。
参见图3和4,通道层200与微井阵列层100层叠设置,通道层上设置有凹槽210,大微井110和小微井120的开口朝向凹槽210。由于微井阵列层的微井和凹槽预先经过表面等离子处理,两者可键合成为一体,从而避免加入液体发生泄漏,向凹槽内加入液滴,由于液滴比油要轻,故在浮力的作用下,微井可捕获液滴。
根据本发明的实施例,凹槽210与大微井110或小微井120通过化学键相连。由此,以便于流入凹槽内的液滴可被微井捕获。
具体地,需要加入液滴之前,在微井阵列层上打两个孔,分别为加液口和出液口,两者均与凹槽相对,通过加液口向凹槽中加入液滴,液体将被微井所捕获,然后再通过加液口向凹槽中加入油,以便冲走凹槽中未被捕获的液滴,并从出液口将液滴吸出。
根据本发明的实施例,微井阵列芯片用于实施前面所述筛选药物的方法。前面针对筛选药物的方法所描述的特征和优点,同样适用于该微井阵列芯片,在此不再赘述。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1
步骤一、本实施例采用类似于CN209144161U的液滴生成装置,其中液滴生成芯片更换为专利WO2020063864A1涉及的芯片,注射器更换为BD 30ml注射器,液滴生成示意图如图5所示,参考如下步骤生成细胞液滴、药物液滴及测序磁珠液滴。
1、分子编码的设计
本发明用到的分子编码是基于国际专利WO2021147069A1发明专利中引入的外源序列信息,共有M×N种编码(M=410,N=410)。通过合成该序列作为药物编码,一种条件下的药物对应一种编码。
2、药物液滴的制备
将一定量的分子编码、对应的药物和携带索引序列的索引磁珠混合后注入液滴生成芯 片,生成尺寸均一的分子编码液滴,以此类推生成不同分子编码的液滴。最后将生成的液滴收集混合在同一个收集管中即完成药物液滴的制备。
3、单细胞液滴的制备
将一定量的细胞悬液注入到液滴生成芯片,通过调节细胞浓度,使得液滴中包裹单个细胞的比率较高的同时保证双包率处于较低的水平,再通过细胞分选芯片利用介电泳等方法将生成的液滴进行分选,得到含有单个细胞的液滴。
4、测序磁珠液滴的制备
将一定量的测序磁珠悬液(利用细胞裂解液重悬测序磁珠得到的)注入到液滴生成芯片,通过调节测序磁珠浓度,使得液滴中包裹单个测序磁珠的比率较高的同时保证双包率处于较低的水平,再通过分选芯片利用介电泳等方法将生成的液滴进行分选,得到含有单个测序磁珠的液滴。
步骤二、本实施例利用如图1~4所示的微井芯片捕获液滴,该微井芯片包含微井阵列层和通道层,微井阵列层有28800个连通的微井(孔径分别为90微米和50微米),微井深度为70微米,这样能保证一个大微井只能够捕获住一个大的液滴,一个小微井只能捕获住一个小的液滴,实现了液滴的1:1配对。由于水相的密度低于油相,此时液滴是漂浮在油相上层,为了捕获住液滴则需在实际使用中将芯片翻转180°,使微井口冲下,通过表面等离子处理将其与图2所示的通道层键合,此时通道内含有25000个以上的有效微井。用孔径1.5毫米的打孔器在微井阵列层上打两个孔,分别为加液口和出液口。
具体操作步骤如下:
首先生成包裹单细胞的大液滴(90微米直径),将细胞液滴加载入微井阵列芯片(图6a),让大微井捕获住细胞液滴,如图6b和图7中a所示。
然后将生成多种类型混合的包裹物质、分子编码和索引磁珠的小液滴(50微米直径)加载入微井阵列芯片,让不同类型小液滴随机被小微井捕获,这样每组微井会随机捕获两个物质小液滴和一个单细胞大液滴,待微井捕获住液滴后注入油推走多余液滴,如图6c和图7中b所示,液滴捕获效率在95%以上。
将芯片放置在摇床上轻微摇动让同一组液滴发生碰撞,与此同时用电晕机或者化学试剂全氟丁醇进行液滴融合,如图6d所示,此时融合后的液滴中随机含有一种或两种物质及对应的分子编码,以及索引磁珠(索引序列没有断裂下来,因为没有加断裂试剂),融合使两个小液滴的位置空出来了,融合效率在80%以上。
将液滴孵育一段时间后,加入含有一个测序磁珠的小液滴,这样每组微井会捕获两个测序磁珠液滴,用油将多余液滴推出,此时的液滴状态如图6e所示。
最后将液滴融合后,由于测序磁珠液滴中含有细胞裂解液和索引序列断裂试剂,这样会使细胞发生裂解,与此同时断裂试剂会将索引磁珠上的索引序列断裂下来。这样测序磁珠会捕获细胞裂解产生的mRNA、索引磁珠断裂下来的索引序列、物质对应的分子编码。
步骤三、液滴孵育完成后,将微流控芯片取出,接着注入测序磁珠液滴,此时该液滴会再次被空出来的小微井捕获,然后用油将多余液滴推出,此时液滴的状态如图6的e所示。最后重复步骤二液滴融合的过程,此时液滴的状态如图6的f所示。
步骤四、为了回收二次融合后的液滴,需要将微井阵列芯片进行翻转180°,接着用油将从微井中脱离的液滴推出芯片,收集液滴于离心管中。如图8所示由于测序磁珠液滴中含有细胞裂解液,一旦液滴二次融合后,细胞会被裂解释放出细胞内的mRNA,此时测序磁珠会捕获包括mRNA、分子编码和索引序列在内的所有核酸信息。最后进行华大单细胞测序流程,通过生物信息学分析将标记同种类索引序列的磁珠归类为来源于同一个液滴。其中将分别纯化二次特异性扩增索引序列和二次特异性扩增物质编码的反应液进行跑胶验证,其实验结果如图9和图10所示,分别在170bp和130bp左右处有明显的峰,证明该发明能够通过单细胞测序技术进行组合物质分子编码检测,索引序列的引入是为了标记同一个液滴中多个测序磁珠。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (15)

  1. 一种确定多种物质与细胞作用关系的方法,其特征在于,包括:
    (1)提供第一液滴、第二液滴、第三液滴和微井阵列芯片;
    其中,所述第一液滴为含有多个不同分子编码液滴的混合液滴,所述第二液滴为含有单个细胞的液滴,所述第三液滴为含有单个测序磁珠、细胞裂解液和断裂试剂的液滴,每个所述分子编码液滴中含有一种物质及与其匹配的编码核酸分子和索引磁珠,所述微井阵列芯上有多个微井组合,每个所述微井组合包括一个大微井和多个与所述大微井相邻且连通的小微井,所述大微井的孔径大于小微井的孔径;
    (2)先将所述第二液滴加入所述微井阵列芯片内并落入大微井内,再将所述第一液滴加入所述微井阵列芯片内并落入多个小微井内;
    (3)使所述第一液滴和第二液滴发生融合,得到第一融合液滴,所述第一融合液滴占据在所述大微井内,所述小微井空出,将所述微井阵列芯片进行细胞培养;
    (4)所述细胞培养完成后,将所述第三液滴加入所述空出的小微井内,将所述第三液滴与所述细胞培养后的第一融合液滴发生融合,在所述第三液滴中的细胞裂解液作用下发生细胞破裂,细胞内的核酸分子与所述编码核酸分子一同被测序磁珠所捕获,同时,在所述第三液滴中的断裂试剂作用下所述索引磁珠上的索引序列将断裂下来,也被测序磁珠所捕获,得到第二融合液滴,收集所述第二融合液滴;
    (5)将所述第二融合液滴进行破乳,收集测序磁珠,将所述测序磁珠上携带的核酸和索引序列进行建库和测序,基于测序结果确定多种物质与细胞作用关系。
  2. 根据权利要求1所述的方法,其特征在于,每个所述分子编码液滴中含有3~8个索引磁珠,每个所述索引磁珠上含有的索引序列不同。
  3. 根据权利要求1或2所述的方法,其特征在于,所述断裂试剂适于断裂二硫键。
  4. 根据权利要求1~3任一项所述的方法,其特征在于,所述断裂试剂选自二硫苏糖醇、三(2-羰基乙基)磷盐酸盐、三(3-羟基丙基)膦和/或β-硫基乙醇。
  5. 根据权利要求1~4任一项所述的方法,其特征在于,所述第二液滴和第三液滴通过分选芯片进行分选得到。
  6. 根据权利要求1~5任一项所述的方法,其特征在于,所述融合为电融合或者化学融合。
  7. 根据权利要求1~6任一项所述的方法,其特征在于,所述大微井的孔径为80~100微米,深度为60~80微米;
    所述小微井的孔径为40~60微米,深度为60~80微米。
  8. 根据权利要求1~7任一项所述的方法,其特征在于,所述测序磁珠适于捕获核酸分子和索引序列。
  9. 根据权利要求1~8任一项所述的方法,其特征在于,进行步骤(2)之前,将步骤(1)所提供的微井阵列芯片进行表面等离子处理,以使所述微井阵列芯片中供液滴流动的凹槽与所述大微井和小微井发生键合,以便于捕获液滴。
  10. 根据权利要求1~9任一项所述的方法,其特征在于,所述收集第二融合液滴的方法包括:
    将所述微井阵列芯片翻转180°,以使所述大微井和小微井的开口向上,向所述微井阵列芯片中加入油,以使所述第二融合液滴从所述大微井内流出至收集容器中。
  11. 一种微井阵列芯片,其特征在于,包括:
    微井阵列层,所述微井阵列层上设置有多个微井组合,每个所述微井组合包括一个大微井和多个与所述大微井相邻且连通的小微井,所述大微井的孔径大于小微井的孔径;
    通道层,所述通道层与所述微井阵列层层叠设置,所述通道层上设置有凹槽,所述大微井和小微井的开口朝向所述凹槽。
  12. 根据权利要求11所述的微井阵列芯片,其特征在于,所述大微井的孔径为80~100微米,深度为60~80微米。
  13. 根据权利要求11或12所述的微井阵列芯片,其特征在于,所述小微井的孔径为40~60微米,深度为60~80微米。
  14. 根据权利要求11~13任一项所述的微井阵列芯片,其特征在于,所述凹槽与所述大微井或小微井通过化学键相连。
  15. 根据权利要求11~14任一项所述的微井阵列芯片,其特征在于,所述微井阵列芯片用于实施权利要求1~10任一项所述的方法。
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