WO2020134612A1 - Microsphère d'hydrogel soluble, son procédé de préparation et son utilisation dans la détection de cellules individuelles - Google Patents

Microsphère d'hydrogel soluble, son procédé de préparation et son utilisation dans la détection de cellules individuelles Download PDF

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WO2020134612A1
WO2020134612A1 PCT/CN2019/115833 CN2019115833W WO2020134612A1 WO 2020134612 A1 WO2020134612 A1 WO 2020134612A1 CN 2019115833 W CN2019115833 W CN 2019115833W WO 2020134612 A1 WO2020134612 A1 WO 2020134612A1
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single cell
soluble hydrogel
soluble
microspheres
droplets
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PCT/CN2019/115833
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张惠丹
李莹玉
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苏州绘真生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/36—Amides or imides
    • C08F222/38—Amides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/24—Homopolymers or copolymers of amides or imides
    • C08L33/26—Homopolymers or copolymers of acrylamide or methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L83/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon only; Compositions of derivatives of such polymers
    • C08L83/04—Polysiloxanes
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present application relates to a single cell detection method, in particular to a soluble barcode-labeled hydrogel microsphere and a preparation method thereof, and a method for performing single cell detection using droplet microfluidic chip technology and detecting single cell related life information It belongs to the technical field of nucleic acid detection analysis and detection.
  • the single cell sequencing technology aims to amplify and sequence the genome or transcriptome at the single cell level to reflect the genetic information of a single cell. This is because even single cells from the same source are subject to many biological processes and environmental disturbances. There are also differences in terms of what we call cell heterogeneity. Conventional genome sequencing technology cannot avoid the impact of cell heterogeneity. This phenomenon is particularly important in tumor tissue. Tumor tissue is a highly heterogeneous tissue that hides circulating tumor cells in the human circulatory system (circulating tumor cell, CTC) sequencing the whole genome or transcriptome is the most helpful. Information about CTC cells is very important for the diagnosis, monitoring and treatment of diseases.
  • CTC circulating tumor cell
  • Droplet microfluidic technology is the science and technology of generating and manipulating nanoliter to picoscale droplets in a closed microchannel network.
  • the droplet microfluidic system can generate a large number of microreactors in a short time, and each droplet can be used as an independent microreactor, the volume can be as small as picoliters or flying, not only greatly reducing the consumption of samples and reagents, And shorten the reaction time.
  • the microfluidic device based on droplets contains barcoded beads and cells together.
  • a fast, cheap, high-throughput single-cell RNA-seq method was established. This technique isolates the cells in tiny droplets and installs barcode primers for amplification, thereby detecting thousands of cells.
  • Drop-seq can help biologists to further discover and classify human cells, draw cell diversity maps of complex tissues such as the brain, better understand stem cell differentiation, and obtain more genetic information on diseases. Therefore, Drop-seq technology is getting more and more popular. The favor of more researchers has become a hot spot for single cell sequencing.
  • the current implementation method of Drop-seq technology is generally to enclose a single cell suspension sample and barcoded beads and reverse transcriptase (RT) lysate through a microfluidic chip in an oil droplet. After reverse transcription in oil droplets, each single-cell cDNA library is given a unique barcode. Finally, we mixed all single-cell cDNA libraries for sequencing, and then identified the barcodes through the program to distinguish single cells.
  • RT reverse transcriptase
  • the first method is to use plastic beads as microbeads carrying barcodes, and use a microfluidic device to wrap a single cell suspension, plastic beads with barcodes, and lysate reverse transcriptase (RT) mixture in a droplet micro reaction In the device.
  • RT reverse transcriptase
  • single cells are lysed, and genetic material such as RNA is released from the lysed cells and is specifically captured by the barcode to form a specific single strand.
  • the reverse transcriptase the reverse transcription reaction is specifically catalyzed to amplify cDNA.
  • the main problem of this method is the poor plasticity of the plastic beads, which leads to blockage of the channel during the formation of droplets, thereby reducing the capture rate of single cells.
  • the second method uses hydrogel microspheres as barcode-carrying microbeads, using a microfluidic device to wrap a single cell suspension, a barcoded hydrogel, and a lysate reverse transcriptase (RT) mixture in one solution Drop in the microreactor. Under the action of the lysate, single cells are lysed, genetic material such as RNA is released from the lysed cells, and is specifically captured by the barcode. Under the action of reverse transcriptase, it specifically catalyzes the reverse transcription reaction. The entire reaction process is Performed in a single droplet microreactor.
  • RT reverse transcriptase
  • the single cell is not encapsulated in the droplet microreactor because the cDNA amplification reaction process is performed in a single droplet microreactor
  • the reverse transcriptase of the suspension did not play a role, which reduced the utilization of reverse transcriptase to a certain extent.
  • barcode-labeled hydrogel microspheres occupy an important position in single-cell detection. It can classify different cells and analyze single-cell information through later bioinformatics techniques to distinguish them. Most of the prior art hydrogel microspheres are insoluble, and cDNA amplification is performed on the gel microspheres, and there are certain limitations to the amount and effect of cDNA amplification.
  • the main purpose of the present application is to provide a soluble nucleic acid barcode-labeled hydrogel microsphere and a preparation method thereof to overcome the deficiencies in the prior art.
  • Another object of the present application is also to provide the application of the soluble hydrogel microspheres in single cell detection.
  • Another object of the present application is to provide a single cell detection method based on droplet microfluidic technology to overcome the deficiencies in the prior art.
  • the embodiments of the present application provide a method for preparing a soluble hydrogel microsphere, which includes:
  • a single emulsion device is used to prepare the microdroplets with the prepolymer solution as the internal phase and the oil phase material as the external phase, and then online polymerization is performed at 30°C to 65°C for 10 to 14 hours to obtain soluble hydrogel microspheres.
  • the examples of the present application also provide the application of the soluble hydrogel microspheres prepared by the foregoing method in the detection of single cells.
  • An embodiment of the present application provides a single cell detection method based on droplet microfluidic technology, which includes:
  • soluble hydrogel microspheres prepared by the foregoing method, and mark with a barcode (referred to as barcode) to obtain soluble hydrogel microspheres with barcode labeling;
  • a droplet microfluidic device is used to inject a single cell suspension, soluble hydrogel microspheres with bar code labeling, reverse transcriptase lysate and oil phase material into the droplet microreactor and form a water-in-oil solution Droplets, wherein the droplets contain at least one single cell and at least one soluble hydrogel microsphere with a barcode label, and the barcode on the hydrogel microsphere can specifically capture the single cell and release it after lysis Genetic material
  • the droplet contains a single cell and a single soluble hydrogel microsphere with a barcode label.
  • the preparation method of soluble nucleic acid barcode-labeled hydrogel microspheres provided in this application is applied to single cell detection based on microfluidic chip counting.
  • the single cell detection method breaks droplets before reverse transcription, which can improve reverse transcription
  • the utilization rate of enzymes, and the use of soluble hydrogel microspheres with barcode labels can prevent channel clogging, which is a method that can effectively improve the utilization rate of reverse transcriptase and solve the channel clogging, which solves the existing technology
  • the plastic bar code carrier is used in the droplet to perform cDNA amplification, resulting in problems such as low ability to capture single cells and unsatisfactory cDNA amplification effect;
  • This application uses droplet microfluidic technology for single cell detection, which solves the problems of difficult operation, cumbersomeness, high cost and low throughput, and has a wide range of application prospects.
  • FIGS 1 and 2 are schematic diagrams of the droplet generation process and the situation in which the hydrogel microspheres are wrapped by droplets in Example 1 of the present application.
  • FIG. 3 is a schematic diagram of a single cell coating after fluorescent staining in Example 1 of the present application.
  • FIGS 4 and 5 are schematic diagrams of the droplet generation process and the situation in which the hydrogel microspheres are wrapped by droplets in Example 2 of the present application.
  • FIG. 6 is a schematic diagram of the coating situation of single cells after fluorescent staining in Example 2 of the present application.
  • FIG 7 and 8 are schematic diagrams of the droplet generation process and the situation in which the hydrogel microspheres are wrapped by droplets in Example 3 of the present application.
  • An aspect of an embodiment of the present application provides a method for preparing soluble hydrogel microspheres, which includes:
  • a single emulsion device is used to prepare the microdroplets with the prepolymer solution as the internal phase and the oil phase material as the external phase, and then online polymerization is performed at 30°C to 65°C for 10 to 14 hours to obtain soluble hydrogel microspheres.
  • the flow rate of the prepolymer solution is 200-400 ⁇ L/h
  • the flow rate of the oil phase material is 400-600 ⁇ L/h
  • an appropriate flow rate is selected according to the required microsphere size.
  • the diameter of the soluble hydrogel microspheres is 20-120 ⁇ m.
  • oil phase substance includes silicone oil (viscosity 10 cSt), perfluorotributylamine (FC-40), other perfluoro oils, etc., but is not limited thereto.
  • the mass ratio of the acrylamide to N,N-bis(acryl)amide is 15:1 to 29:1.
  • the volume ratio of the 2-hydroxy-2-methyl-1-phenyl-1-acetone to the mixed mother liquor is 10-50 ⁇ L: 1 mL.
  • the added amount of 2-hydroxy-2-methyl-1-phenyl-1-acetone is related to the external reaction temperature.
  • the preparation method further includes: collecting the obtained soluble hydrogel microspheres, and washing to remove excess oil phase substances, and then replacing with a gradient of ethanol aqueous solution to collect the soluble hydrogel microspheres In water.
  • the preparation method specifically includes the following steps:
  • microdroplets are prepared by a single emulsion device, and are polymerized online to obtain soluble hydrogel microspheres.
  • the size of the generated hydrogel microspheres can be controlled.
  • Another aspect of the embodiments of the present application also provides the application of the soluble hydrogel microspheres prepared by the foregoing method in single cell detection.
  • Another aspect of the embodiments of the present application also provides a single cell detection method based on droplet microfluidic technology, which includes:
  • soluble hydrogel microspheres prepared by the foregoing method, and mark with a barcode (referred to as barcode) to obtain soluble hydrogel microspheres with barcode labeling;
  • a droplet microfluidic device is used to inject a single cell suspension, soluble hydrogel microspheres with bar code labeling, reverse transcriptase lysate and oil phase material into the droplet microreactor and form a water-in-oil solution Droplets, wherein the droplets contain at least one single cell and at least one soluble hydrogel microsphere with a barcode label, and the barcode on the soluble hydrogel microsphere can specifically capture the single cell and release it after lysis Genetic material
  • the single cell detection method specifically includes: dissolving the soluble hydrogel microspheres at room temperature using a mixture of dimercaptothreitol solution, ⁇ -mercaptoethanol and ⁇ -mercaptoethanol .
  • the method includes: dissolving dithiothreitol with an acetate-sodium acetate buffer to obtain a dithiothreitol solution.
  • dimercaptothreitol is dissolved in 0.01 Mol/L pH 5.2 acetic acid-sodium acetate buffer to obtain 1-10 mmol dimercaptothreitol solution, which is stored at -20°C for later use.
  • dimercaptothreitol solution adjusts the pH to alkaline with sodium hydroxide, and immerse the hydrogel microspheres in the solution. After a period of time (5-30min), soluble hydrogel microspheres degraded in solution.
  • the process of dissolving the soluble hydrogel microspheres includes: dimercaptothreitol (DTT), ⁇ -mercaptoethanol or ⁇ -mercaptoethanol and the like can be used to dissolve the soluble hydrogel microspheres Ball, in which the DTT concentration is between 1 ⁇ 10mmol/L, according to different experimental requirements, choose different concentrations, the concentration range of ⁇ -mercaptoethanol and ⁇ -mercaptoethanol is 0.1 ⁇ 1wt%. In between, placing at room temperature can fully dissolve soluble hydrogel microspheres.
  • DTT dimercaptothreitol
  • ⁇ -mercaptoethanol or ⁇ -mercaptoethanol a concentration range of ⁇ -mercaptoethanol and ⁇ -mercaptoethanol
  • the droplet in the single-cell detection method, is a water-in-oil structure.
  • Changing the surface property can break the droplet and release the reactant in the droplet. Therefore, to break the droplets, alternative methods include physical methods and chemical methods.
  • the physical method may be a temperature change method, a heating method, or an energization method. Without changing the temperature of the droplets, the droplets can be transferred to a low temperature of -20 °C and placed for 20 minutes, and then transferred to room temperature for 5 minutes. Repeat this 3 times to break the droplets; heating method, the temperature is higher than 100 °C, more than 10 minutes , Can break the droplets; current, high voltage 220-380V, 30S can break the droplets.
  • the single cell detection method specifically includes: injecting a single cell suspension, a soluble hydrogel microsphere with a barcode label, a reverse transcriptase lysate, and an oily phase substance into a droplet by means of a syringe pump or a vacuum pump
  • the microfluidic chip channel of the microreactor forms water-in-oil droplets.
  • the droplet contains a single cell and a single soluble hydrogel microsphere with a barcode label.
  • the diameter of the soluble hydrogel microspheres is 20-120 ⁇ m, and the larger the diameter, the easier it is to break.
  • the soluble hydrogel microspheres include polyacrylamide hydrogel microspheres.
  • the single cell detection method specifically includes: marking a barcode on the soluble hydrogel microspheres to obtain a soluble hydrogel microsphere with a barcode label.
  • the specific process of labeling is: fixing a primer on the hydrogel microspheres, using isothermal amplification enzymes, and connecting 96 different Oligo primers to the hydrogel microspheres respectively, to obtain 96 kinds of Oligo libraries, using the same method, 96 kinds of additional Oligo up connection, a short nucleotide finally obtained about 104 sized for Oligo libraries.
  • the single cell suspension includes any one or a combination of two or more of bacterial suspension, fungal suspension, animal cell suspension, etc., but is not limited thereto.
  • oil phase substance includes perfluorotributylamine perfluorooil phase components, surfactants, and the like.
  • the surfactant includes Span 80, Tween 20, etc., but is not limited thereto.
  • the genetic material released after the single cell is lysed includes DNA, RNA, etc., but is not limited thereto.
  • this application uses soluble hydrogel microspheres as barcode-carrying microbeads, using a microfluidic device to separate single cell suspensions, barcoded soluble hydrogel microspheres, and lysate reverse transcriptase (RT )
  • the mixed solution is wrapped in a droplet microreactor. Under the action of the lysate, single cells are lysed, and genetic material such as RNA is released from the lysed cells, specifically captured by the barcode on the hydrogel microspheres, and then the droplets are broken up and the soluble hydrogel microspheres are dissolved Under the action of reverse transcriptase, it specifically catalyzes the reverse transcription reaction to perform cDNA amplification.
  • the method for preparing soluble hydrogel microspheres labeled with soluble nucleic acid barcodes is applied to single cell detection based on microfluidic chip counting, which breaks droplets before reverse transcription , Can improve the utilization rate of reverse transcriptase, and can prevent channel blockage by using soluble hydrogel microspheres with barcode labeling, which is a method that can effectively improve the utilization rate of reverse transcriptase and solve the channel blockage, It solves the problems of using plastic barcode carrier and cDNA amplification in droplets in the prior art, resulting in low ability to capture single cells and unsatisfactory cDNA amplification effect.
  • the single-cell detection method based on microfluidics and the preparation method of soluble nucleic acid barcode-labeled hydrogel microspheres of the present application can be used in various fields of single-cell detection, for example: for sequencing single-cell transcriptomes of tumor cells and for single-cell immune cells Cell transcriptome sequencing, etc.
  • microdroplets are prepared by a single emulsion device.
  • buffer solution 250Mm Tris-Hcl, 375mMol/L KCl, 15mMol/L MgCl 2
  • RT-lysis lysate 50 ⁇ l 2x lysis buffer (5x RT buffer 20 ⁇ l, enzyme 5 ⁇ l, nucleic acid primer polyadenylation tail 2 ⁇ l, dNTP 4 ⁇ l, 10% NP40 10 ⁇ l, dH) 2 O 9 ⁇ l), put on ice for use.
  • microdroplets are prepared by a single emulsion device.
  • buffer solution 250Mm Tris-Hcl, 375mMol/L KCl, 15mMol/L MgCl 2
  • RT-lysis lysate 50 ⁇ l 2x lysis buffer (5x RT buffer 20 ⁇ l, enzyme 5 ⁇ l, nucleic acid primer polyadenylation tail 2 ⁇ l, dNTP 4 ⁇ l, 10% NP40 10 ⁇ l, dH) 2 O 9 ⁇ l), put on ice for use.
  • microdroplets are prepared by a single emulsion device.
  • RT-lysis lysate 50 ⁇ l 2x lysis buffer (5x RT buffer 20 ⁇ l, enzyme 5 ⁇ l, nucleic acid primer polyadenylation tail 2 ⁇ l, dNTP 4 ⁇ l, 10% NP40 10 ⁇ l, dH) 2 O 9 ⁇ l), put on ice for use.
  • a single cell detection method based on droplet microfluidic technology mainly includes the following steps:
  • 1RT lysate NaCl, NP-40, Tween-20, SDS, EDTA, PMSF, Tris-HCl, Aprotinin, Leupeptin, Sodium dexycholate.
  • microdroplets are prepared by a single emulsion device.
  • Bacterial suspension pick a single bacterium and settle it in the liquid medium for cultivation overnight, measure the OD value of bacterial growth, dilute according to the experimental requirements and obtain the corresponding volume of bacterial suspension;
  • Animal cell suspension Take animal tissue and obtain a single cell suspension of animal tissue by mechanical, chemical or enzymatic digestion;
  • Oil phase preparation perfluorotributylamine (FC-40) perfluoro oil phase, add the corresponding surfactant Span 80, Tween-20.
  • Barcode-labeled soluble hydrogel microspheres capture DNA or RNA substances released after single cell lysis
  • the droplets were transferred to a low temperature of -20°C for 20 minutes, and then transferred to room temperature for 5 minutes. This was repeated 3 times to break the droplets.
  • nucleic acid barcode-labeled soluble hydrogel microspheres 250Mm Tris-Hcl, 375mMKCl, 15mM MgCl 2 ) wash the hydrogel microspheres, draw 25-30 ⁇ l of nucleic acid barcode-labeled soluble hydrogel microspheres to In a 1.5ml EP tube, add 1ml of buffer, room temperature, 5000g, 1min. Discard the upper buffer as much as possible.
  • RT-lysis lysate 50 ⁇ l of 2x Lysis buffer (5x RTbuffer 20 ⁇ l, enzyme 5 ⁇ l, OligodT 2 ⁇ l, dNTP 4 ⁇ l, 10% NP4010 ⁇ l, dH2O 9 ⁇ l) are placed on ice in the ultra-clean workbench.
  • the preparation method of nucleic acid barcode-labeled soluble hydrogel microspheres provided by the present application is applied to single-cell detection based on microfluidic chip counting.
  • the single-cell detection method is used in reverse transcription Breaking the droplets before can improve the utilization rate of reverse transcriptase, and by using soluble hydrogel microspheres with barcode labels to prevent channel blockage, it is a way to effectively improve the utilization rate of reverse transcriptase and solve
  • the method of channel clogging solves the problems of using plastic barcode carriers and cDNA amplification in droplets in the prior art, resulting in low ability to capture single cells and unsatisfactory cDNA amplification effects.
  • Examples 1 to 5 the inventors of the present case also conducted experiments with other materials and conditions listed in this specification by referring to Examples 1 to 5, and also prepared soluble hydrogel microspheres labeled with nucleic acid barcodes, and these The performance of soluble hydrogel microspheres labeled with nucleic acid barcodes is basically the same as that in Examples 1-5.

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Abstract

La présente invention concerne une microsphère d'hydrogel, son procédé de préparation et son utilisation dans la détection de cellules individuelles. Le procédé de préparation comprend: le mélange uniforme d'acrylamide, de N, N-bis (acrolyl)amide, d'acide acétique et de 2-hydroxyl-2-méthyl-1-phényl-1-acétone, et l'ajout d'hydroxyde de sodium dans celui-ci pour obtenir une solution de prépolymère; l'utilisation d'un dispositif d'émulsion unique et l'utilisation de la solution de prépolymère en tant que phase interne et d'une substance de phase huileuse en tant que phase externe pour obtenir des microgouttelettes; et la réalisation d'une polymérisation en ligne sur celles-ci pour obtenir la microsphère. Le procédé de détection de cellule individuelle comprend: le marquage d'un code à barres sur une microsphère d'hydrogel soluble; l'injection de la microsphère d'hydrogel soluble, d'une suspension de cellule individuelle et d'un lysat de transcriptase inverse dans un micro-réacteur à gouttelettes pour former des gouttelettes d'eau dans l'huile, la rupture des gouttelettes et la dissolution de la microsphère d'hydrogel soluble, et la réalisation d'une réaction transcriptionnelle inverse catalytique spécifique et d'une amplification d'ADNc; et la distinction de la cellule individuelle au moyen de la reconnaissance du code à barres. Le procédé de détection de cellule individuelle peut non seulement améliorer efficacement le taux d'utilisation de la transcriptase inverse, mais peut également résoudre le problème d'obstruction de canal.
PCT/CN2019/115833 2018-12-29 2019-11-06 Microsphère d'hydrogel soluble, son procédé de préparation et son utilisation dans la détection de cellules individuelles WO2020134612A1 (fr)

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CN110305941A (zh) * 2019-06-21 2019-10-08 苏州锐讯生物科技有限公司 一种基于微流控技术获取单样本遗传信息的方法
CN110302726A (zh) * 2019-07-30 2019-10-08 苏州济研生物医药科技有限公司 一种基于微流控的负载细胞水凝胶微珠制备装置及方法
WO2021128036A1 (fr) * 2019-12-25 2021-07-01 苏州绘真生物科技有限公司 Kit permettant de construire une bibliothèque de séquençage de tcr monocellulaire humain et son utilisation
CN111172257A (zh) * 2020-01-16 2020-05-19 南方科技大学 一种带编码的凝胶微粒及其制备方法和应用
CN111500440A (zh) * 2020-04-26 2020-08-07 中国科学院广州生物医药与健康研究院 一种单细胞分选装置和单细胞分选方法
CN112251504A (zh) * 2020-09-09 2021-01-22 新格元(南京)生物科技有限公司 一种具有分子标签序列的磁性微球及其制备方法
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