JPWO2020161500A5 - - Google Patents

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JPWO2020161500A5
JPWO2020161500A5 JP2021546352A JP2021546352A JPWO2020161500A5 JP WO2020161500 A5 JPWO2020161500 A5 JP WO2020161500A5 JP 2021546352 A JP2021546352 A JP 2021546352A JP 2021546352 A JP2021546352 A JP 2021546352A JP WO2020161500 A5 JPWO2020161500 A5 JP WO2020161500A5
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microdroplets
average volume
volume
aqueous medium
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JP2022519742A (en
JP7314290B2 (en
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微小液滴のサイズを制御する、かつ／又は、微小液滴で生じる酵素反応又は化学反応を維持若しくは最適化する方法であり、前記微小液滴は、少なくとも１つの生物学的成分と、１未満の水分活性ａｗ１を有する第１の水性媒体とを含み、０．５フェムトリットルから１０ナノリットルの範囲の平均体積を有し、前記微小液滴を、第２の水性媒体を含み、前記微小液滴の平均体積の２５％未満の平均体積を有し、最大４フェムトリットルまでの二次液滴をさらに含む水非混和性の担体液中に維持するステップを特徴とし、全体の単位体積あたりの微小液滴の合計体積に対する担体液の体積の比が２：１より大きい、方法。 1. A method of controlling the size of microdroplets and/or maintaining or optimizing enzymatic or chemical reactions occurring in microdroplets, said microdroplets comprising at least one biological component and less than one and having an average volume in the range of 0.5 femtoliter to 10 nanoliters, said microdroplets comprising a second aqueous medium, said microdroplets comprising maintained in a water-immiscible carrier liquid further comprising up to 4 femtoliters of secondary droplets having an average volume of less than 25% of the average volume of the droplets, and wherein the ratio of the volume of the carrier liquid to the total volume of the microdroplets of is greater than 2:1. ０．５フェムトリットルから１０ナノリットルの範囲の平均体積を有する微小液滴の内容物のサイズ及び／又は化学反応性若しくは酵素反応性を制御する方法であり、前記微小液滴は、少なくとも１つの生物学的成分と、１未満の水分活性ａｗ１を有する生物学的細胞を含まない第１の水性媒体とを含み、前記微小液滴を、第２の水性媒体を含み、前記微小液滴の平均体積の２５％未満の平均体積を有し、最大０．５フェムトリットルまでの二次液滴をさらに含む水非混和性の担体液中に維持するステップを特徴とし、全体の単位体積あたりの微小液滴の合計体積に対する担体液の体積の比が２：１より大きい、請求項１に記載の方法。 A method of controlling the size and/or chemical or enzymatic reactivity of the contents of microdroplets having an average volume in the range of 0.5 femtoliter to 10 nanoliters, said microdroplets comprising at least one a biological component and a first aqueous medium free of biological cells having a water activity a w1 of less than 1; said microdroplets comprising a second aqueous medium; maintained in a water-immiscible carrier liquid having an average volume of less than 25% of the average volume and further comprising up to 0.5 femtoliters of secondary droplets, 2. The method of claim 1, wherein the ratio of the volume of carrier liquid to the total volume of microdroplets is greater than 2:1. ４フェムトリットルから１０ナノリットルの範囲の平均体積を有する微小液滴における化学反応性若しくは酵素反応性及び／又は微小液滴サイズを制御する方法であり、前記微小液滴は、少なくとも１つの生物学的細胞と、１未満の水分活性ａｗ１を有する第１の水性媒体とを含み、前記微小液滴を、第２の水性媒体を含み、前記微小液滴の平均体積の２５％未満の平均体積を有し、最大４フェムトリットルまでの二次液滴をさらに含む水非混和性の担体液中に維持するステップを特徴とし、全体の単位体積あたりの微小液滴の合計体積に対する担体液の体積の比が２：１より大きい、請求項１に記載の方法。 1. A method of controlling chemical or enzymatic reactivity and/or microdroplet size in microdroplets having an average volume ranging from 4 femtoliters to 10 nanoliters, said microdroplets comprising at least one biological and a first aqueous medium having a water activity a w1 of less than 1, said microdroplets comprising a second aqueous medium , having an average volume of less than 25% of the average volume of said microdroplets. and maintaining in a water-immiscible carrier liquid further comprising up to 4 femtoliters of secondary droplets, the volume of carrier liquid relative to the total volume of microdroplets per unit volume of the whole The method of claim 1, wherein the ratio of is greater than 2:1. 前記二次液滴がａｗ１よりも大きい水分活性ａｗ２を有することを特徴とする、請求項１～３のいずれか一項に記載の方法。 A method according to any one of claims 1 to 3, characterized in that said secondary droplets have a water activity a w2 greater than a w1 . 前記二次液滴がａｗ１よりも小さい水分活性ａｗ２を有することを特徴とする、請求項１～３のいずれか一項に記載の方法。 A method according to any one of claims 1 to 3, characterized in that said secondary droplets have a water activity a w2 smaller than a w1 . 前記水分活性ａｗ１及びａｗ２が同じであることを特徴とする、請求項１～３のいずれか一項に記載の方法。 Process according to any one of claims 1 to 3, characterized in that the water activities a w1 and a w2 are the same. ａｗ１及びａｗ２が独立して０．９～１の範囲にあることを特徴とする、請求項１～６のいずれか一項に記載の方法。 A method according to any one of claims 1 to 6, characterized in that a w1 and a w2 are independently in the range 0.9-1. 前記第２の水性媒体のイオン強度が、前記第１の水性媒体のイオン強度の１～５倍の範囲にあることを特徴とする、請求項４に記載の方法。 A method according to claim 4, characterized in that the ionic strength of said second aqueous medium is in the range of 1-5 times the ionic strength of said first aqueous medium. 前記第１の媒体のイオン強度が、前記第２の水性媒体のイオン強度の１～５倍の範囲にあることを特徴とする、請求項５に記載の方法。 A method according to claim 5, characterized in that the ionic strength of said first medium is in the range of 1 to 5 times the ionic strength of said second aqueous medium. 前記二次液滴の平均体積が前記微小液滴の平均体積の１０％未満であることを特徴とする、請求項１～９のいずれか一項に記載の方法。 Method according to any one of the preceding claims, characterized in that the average volume of the secondary droplets is less than 10% of the average volume of the microdroplets. 前記第１及び第２の水性媒体のうちの少なくとも１つがさらにグリセロールを含むことを特徴とする、請求項１～１０のいずれか一項に記載の方法。 Method according to any one of the preceding claims, characterized in that at least one of said first and second aqueous media further comprises glycerol. 前記生物学的成分が、標的核酸に由来する単一ヌクレオシド三リン酸、細胞のＤＮＡ若しくはＲＮＡに由来するオリゴヌクレオチド、酵素、又は細胞から選択されることを特徴とする、請求項１～１１のいずれか一項に記載の方法。 of claims 1 to 11, characterized in that said biological component is selected from single nucleoside triphosphates derived from target nucleic acids, oligonucleotides derived from cellular DNA or RNA, enzymes or cells. A method according to any one of paragraphs. 第１及び／又は第２の水性媒体が緩衝液であることを特徴とする、請求項１２に記載の方法。 13. Method according to claim 12, characterized in that the first and/or second aqueous medium is a buffer. ４フェムトリットルから１０ナノリットルの範囲の平均体積を有する微小液滴内に含まれる１つ又は複数の細胞型の細胞増殖を、前記液滴内の細胞を適切な環境条件でインキュベートし、その後、各液滴内の細胞数を検出することにより引き起こすことを更に含む、請求項３に記載の方法。 Cell growth of one or more cell types contained within microdroplets having an average volume in the range of 4 femtoliters to 10 nanoliters by incubating the cells within said droplets in suitable environmental conditions, followed by , by detecting the number of cells in each droplet . 検討中の細胞の１つ又は複数の表現型形質、遺伝形質、又はタンパク質発現プロファイルを、前記細胞に由来する標的を標識することにより分析又は検出することを更に含み、前記細胞は、４フェムトリットルから１０ナノリットルの範囲の平均体積を有し、水性緩衝液を含む微小液滴内に含まれる、請求項３に記載の方法。 further comprising analyzing or detecting one or more phenotypic traits, genetic traits, or protein expression profiles of the cells under study by labeling targets derived from said cells , said cells containing 4 femtoliters of 4. The method of claim 3, contained within microdroplets comprising an aqueous buffer, having an average volume in the range of to 10 nanoliters. 核酸分析物をヌクレオシド三リン酸分子の秩序ある流れに加ピロリン酸分解によって次第に消化し、そこから、０．５フェムトリットルから１０ナノリットルの範囲の平均体積を有し、前記ヌクレオシド三リン酸分子の１つと水性緩衝液とを各々含む微小液滴の対応する秩序ある流れを生成することと、各微小液滴内の各ヌクレオシド三リン酸分子と核酸塩基に特異的な蛍光プローブとを反応させ、その後、各微小液滴に関連する対応する蛍光を検出し、それにより前記核酸塩基を同定することにより、配列を決定することを更に含む、請求項２に記載の方法。 Nucleic acid analytes are progressively digested by pyrophosphorolysis into an ordered stream of nucleoside triphosphate molecules from which the nucleoside triphosphates have an average volume ranging from 0.5 femtoliters to 10 nanoliters. generating corresponding ordered streams of microdroplets each containing one of the molecules and an aqueous buffer; and reacting each nucleoside triphosphate molecule within each microdroplet with a nucleobase-specific fluorescent probe. 3. The method of claim 2, further comprising determining the sequence by causing and thereafter detecting the corresponding fluorescence associated with each microdroplet, thereby identifying the nucleobases. 前記微小液滴と前記二次液滴の水分活性の比が０．９：１～１：０．９、好ましくは０．９５：１～１：０．９５の範囲にあることを特徴とする、請求項１４～１６のいずれか一項に記載の方法。
The water activity ratio between the microdroplets and the secondary droplets is in the range of 0.9:1 to 1:0.9, preferably 0.95:1 to 1:0.95. , the method according to any one of claims 14-16.
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