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1. WO2020134612 - SOLUBLE HYDROGEL MICROSPHERE, PREPARATION METHOD THEREFOR, AND USE THEREOF IN SINGLE CELL DETECTION

Publication Number WO/2020/134612
Publication Date 02.07.2020
International Application No. PCT/CN2019/115833
International Filing Date 06.11.2019
IPC
C08F 220/56 2006.1
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
220Copolymers 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
02Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
52Amides or imides
54Amides
56Acrylamide; Methacrylamide
C08F 222/38 2006.1
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
222Copolymers 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
36Amides or imides
38Amides
C08J 3/075 2006.1
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
JWORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H142
3Processes of treating or compounding macromolecular substances
02Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
03in aqueous media
075Macromolecular gels
C08L 33/26 2006.1
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
LCOMPOSITIONS OF MACROMOLECULAR COMPOUNDS
33Compositions 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
24Homopolymers or copolymers of amides or imides
26Homopolymers or copolymers of acrylamide or methacrylamide
C08L 83/04 2006.1
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
LCOMPOSITIONS OF MACROMOLECULAR COMPOUNDS
83Compositions 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
04Polysiloxanes
C12N 11/04 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
11Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
02Enzymes or microbial cells immobilised on or in an organic carrier
04entrapped within the carrier, e.g. gel or hollow fibres
CPC
C08F 220/56
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
220Copolymers 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
02Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
52Amides or imides
54Amides ; , e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
56Acrylamide; Methacrylamide
C08F 222/38
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
222Copolymers 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
36Amides or imides
38Amides
C08J 3/075
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
JWORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G
3Processes of treating or compounding macromolecular substances
02Making solutions, dispersions or lattices by other methods than by solution, emulsion or suspension polymerisation techniques
03in aqueous media
075Macromolecular gels
C08L 33/26
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
LCOMPOSITIONS OF MACROMOLECULAR COMPOUNDS
33Compositions 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
24Homopolymers or copolymers of amides or imides
26Homopolymers or copolymers of acrylamide or methacrylamide
C08L 83/04
CCHEMISTRY; METALLURGY
08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
LCOMPOSITIONS OF MACROMOLECULAR COMPOUNDS
83Compositions 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
04Polysiloxanes
C12N 11/04
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
11Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
02Enzymes or microbial cells immobilised on or in an organic carrier
04entrapped within the carrier, e.g. gel or hollow fibres
Applicants
  • 苏州绘真生物科技有限公司 SUZHOU GENO-TRUTH BIOTECHNOLOGY CO., LTD. [CN]/[CN]
Inventors
  • 张惠丹 ZHANG, Huidan
  • 李莹玉 LI, Yingyu
Agents
  • 南京利丰知识产权代理事务所(特殊普通合伙) NANJING LI & FENG INTELLECTUAL PROPERTY AGENCY (SPECIAL GENERAL PARTNERSHIP)
Priority Data
201811632889.X29.12.2018CN
Publication Language Chinese (ZH)
Filing Language Chinese (ZH)
Designated States
Title
(EN) SOLUBLE HYDROGEL MICROSPHERE, PREPARATION METHOD THEREFOR, AND USE THEREOF IN SINGLE CELL DETECTION
(FR) MICROSPHÈRE D'HYDROGEL SOLUBLE, SON PROCÉDÉ DE PRÉPARATION ET SON UTILISATION DANS LA DÉTECTION DE CELLULES INDIVIDUELLES
(ZH) 可溶性水凝胶微球及其制备方法与在单细胞检测中的应用
Abstract
(EN)
Provided are a soluble hydrogel microsphere, a preparation method therefor, and the use thereof in single cell detection. The preparation method comprises: evenly mixing acrylamide, N,N-bis(acrolyl)amide, acetic acid and 2-hydroxyl-2-methyl-1-phenyl-1-acetone, and adding sodium hydroxide into same to obtain a prepolymer solution; using a single emulsion device and using the prepolymer solution as an internal phase and an oil phase substance as an external phase to obtain micro-droplets; and performing on-line polymerization on same to obtain the microsphere. The single cell detection method comprises: marking a bar code on a soluble hydrogel microsphere; injecting the soluble hydrogel microsphere, a single cell suspension and a reverse transcriptase lysate into a droplet micro-reactor to form water-in-oil droplets, breaking the droplets and dissolving the soluble hydrogel microsphere, and performing a specific catalytic reverse transcriptional reaction and a cDNA amplification; and distinguishing the single cell by means of recognizing the bar code. The single cell detection method can not only effectively improve the utilization rate of the reverse transcriptase, but can also solve the problem of channel obstruction.
(FR)
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.
(ZH)
一种可溶性水凝胶微球及其制备方法与在单细胞检测中的应用。所述制备方法包括:使丙烯酰胺、N,N-双(丙烯酰)酰胺、乙酸和2-羟基-2-甲基-1-苯基-1-丙酮混匀,加入氢氧化钠得到预聚物溶液;采用单乳液装置,以预聚物溶液为内相、油相物质为外相,得到微液滴,在线聚合获得所述微球。所述单细胞检测方法包括:在可溶性水凝胶微球上标记条形码,将其与单细胞悬液、逆转录酶裂解液注入液滴微反应器中形成油包水的液滴,打碎液滴并溶解所述水凝胶微球,进行特异性的催化逆转录反应,以及进行cDNA扩增;通过识别条形码区分单细胞。所述单细胞检测方法既能有效提高逆转录酶的利用率又能解决通道堵塞。
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