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KR-102963903-B1 - Microdroplet Manipulation Method

KR102963903B1KR 102963903 B1KR102963903 B1KR 102963903B1KR-102963903-B1

Abstract

A method for manipulating microdroplets is provided, comprising a first aqueous medium having an average volume in the range of 0.5 femtoliters to 10 nanoliters, at least one biological component, and a water activity a w1 less than 1. The method is characterized by the step of holding the microdroplets in a dispersion medium immiscible with water, further comprising a second droplet comprising a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, wherein the volume ratio of the dispersion medium to the total volume of the microdroplets per total unit volume is greater than 2:1. The method may be used with microdroplets containing, for example, biological cells or microdroplets containing single nucleoside phosphates, such as those produced in droplet-based nucleic acid sequencers. The method is suitable for controlling, for example, cellular, chemical, or enzymatic processes and/or the size of microdroplets in microdroplets or single nucleoside nucleic acid sequencing.

Inventors

  • 아이삭, 톰
  • 밤포스, 바나비
  • 콘테리오, 자스민
  • 존슨, 커 프랜시스
  • 소스나, 마시에지
  • 인검, 리차드
  • 포드, 가레스

Assignees

  • 라이트캐스트 디스커버리 엘티디

Dates

Publication Date
20260511
Application Date
20200207
Priority Date
20190208

Claims (20)

  1. As a method for manipulating microdroplets, The above microdroplet comprises one or more of an oligonucleotide, a fluorescently labeled antibody, a FRET (Fluorescence Resonance Energy Transfer) reporter probe, an enzyme-labeled antigen, a cell lysate, and a cell expression protein, and a first aqueous medium having a water activity a w1 of less than 1, and The above method comprises the step of maintaining the microdroplet in a dispersion medium that is immiscible with water, wherein the dispersion medium further comprises a second droplet comprising a second aqueous medium, and The second droplet has an average volume of less than 25% of the average volume of the microdroplet, and a volume of up to 4 femtoliters, wherein the ratio of the volume of the dispersion medium to the total volume of the microdroplet per total unit volume is greater than 2:1. method.
  2. A method of manipulating a microdroplet comprising at least one biological component and a first aqueous medium having a water activity a w1 less than 1, wherein the average volume is in the range of 0.5 femtoliters to 10 nanoliters. The method is characterized by the step of maintaining a microdroplet in a dispersion medium immiscible with water, further comprising a second droplet comprising a second aqueous medium that is emulsified in the dispersion medium and stabilized by a shell of compatible surfactant molecules. Here, the second droplet has an average volume of less than 25% of the average volume of the microdroplet and reaches a maximum of 4 femtoliters, and the volume ratio of the dispersion medium to the total volume of microdroplets per total unit volume is greater than 2:1. Microdroplet manipulation method.
  3. delete
  4. In claim 2, A microdroplet manipulation method characterized in that the above surfactant is a non-ionic surfactant.
  5. In claim 2, A microdroplet manipulation method characterized in that the above surfactant is a fluorinated surfactant.
  6. In claim 1, A microdroplet manipulation method characterized in that the above dispersion medium is a composite medium.
  7. In claim 1, A microdroplet manipulation method characterized in that the dispersion medium is a hydrated oil.
  8. In claim 1, A microdroplet manipulation method characterized in that the dispersion medium is selected from mineral oil, silicone oil, or fluorocarbon oil.
  9. In claim 1, A microdroplet manipulation method characterized in that the second droplet has a water activity a w2 greater than a w1 .
  10. In claim 1, A microdroplet manipulation method characterized in that the second droplet has a water activity a w2 that is smaller than a w1 .
  11. In any one of claims 1 to 2, A microdroplet manipulation method characterized in that the second droplet has a water activity a w2 equal to a w1 .
  12. In claim 11, A microdroplet manipulation method characterized in that a w1 and a w2 are independently in the range of 0.9 to 1.
  13. In claim 9, A microdroplet manipulation method characterized in that the ionic strength of the second aqueous medium is in the range of 1 to 5 times the ionic strength of the first aqueous medium.
  14. In claim 10, A microdroplet manipulation method characterized in that the ionic strength of the first medium is in the range of 1 to 5 times the ionic strength of the second aqueous medium.
  15. In any one of claims 1 to 2, A microdroplet manipulation method characterized in that the average volume of the second droplet is less than 10% of the average volume of the microdroplet.
  16. In any one of claims 1 to 2, A microdroplet manipulation method characterized in that at least one of the first and second aqueous media further comprises glycerol.
  17. delete
  18. In claim 16, A microdroplet manipulation method characterized in that the first and/or second aqueous medium is a buffer solution.
  19. In claim 2, A microdroplet manipulation method characterized in that the surfactant is a fluorinated surfactant and the dispersion medium is a composite medium containing hydrated fluorocarbon oil.
  20. In claim 1, A microdroplet manipulation method characterized in that the dispersion medium is a composite medium containing hydrated fluorocarbon oil.

Description

Microdroplet Manipulation Method The present invention relates to an improved method for manipulating aqueous microdroplets that selectively contain biological cells in an immiscible dispersion medium such as oil. This enables controlling or adjusting the size of the microdroplets and maintaining or optimizing any enzymatic or chemical reactions occurring therein for a predetermined period. In our previous patent applications, e.g., WO2014167323, WO2015121675, WO2016012789, WO2017140839 and PCT/EP2018/066574, the inventors have described methods for manipulating biological components, such as cells, enzymes, oligonucleotides, and even single nucleotides, for various analytical purposes, including DNA and RNA sequencing analysis, and the detection and characterization analysis of cells and viruses. In some embodiments, these methods involve displacing microdroplets dispersed in an immiscible dispersion medium along a microfluidic path in an analytical device by using an electrowetting propulsion force or by directly discharging microdroplets onto a substrate coated with a dispersion medium. It has been found that in many cases where the volume fraction of the microdroplets is relatively low, these microdroplets tend to shrink significantly over time, which can sometimes interfere with some or all enzymatic processes proceeding within them. In addition, in other cases, it may be desirable to intentionally shrink or increase the size of microdroplets in a part of the device as a predetermined analysis is performed. Figure 1 compares the results obtained after 24 hours of incubation with reference measurements at 0 and 24 hours of unhydrated oil. The data presented as a histogram in Figure 2 shows the average fluorescence intensity of 6 μm droplets cultured in unhydrated oil ('Dry oil'), oil hydrated with water ('Water only'), or oil hydrated to three times the concentration of the droplet buffer ('3x buffer'). The inventors have developed a method for manipulating microdroplets to overcome these problems. This method can be used, for example, to manipulate the size and/or reactivity of the contents of microdroplets or to control chemical or enzymatic reactions occurring therein. The invention is defined in the appended claims. According to one aspect of the invention, a general method for manipulating (controlling the size and/or chemical composition of the contents of microdroplets) a microdroplet comprising at least one biological component and a first aqueous medium having an average volume in the range of 0.5 femtoliters to 10 nanoliters and a water activity a w1 less than 1 is provided, characterized by the step of holding the microdroplet in a dispersion medium that is immiscible with water, further comprising a second droplet comprising a second aqueous medium having an average volume less than 25% of the average volume of the microdroplet and up to 4 femtoliters, wherein the volume ratio of the dispersion medium to the total volume of the microdroplet per total unit volume is greater than 2:1. Although not intended to limit the scope of the invention, it is believed that the invention aims to solve the problem by using a dispersion medium containing very small second droplets capable of interacting with microdroplets without adversely affecting the overall characteristics of the microdroplets or the efficacy of any detection method applied to them. When the dispersion medium is an oil, such a composite medium is sometimes referred to as a 'hydrated oil'. An important feature in this regard is that the relative water activity of the microdroplets and the second droplets is controlled within specific variables, optionally by continuous monitoring and/or a feedback loop. Here, the water activity of the aqueous medium (a w ) is defined as the ratio of the partial vapor pressure of the aqueous medium under investigation to the partial vapor pressure of pure water under STP conditions. Since water tends to diffuse along a gradient of high water activity to low water activity, it was confirmed that when the water activity of the second aqueous medium (a w2 ) is higher than the water activity of the first aqueous medium (a w1 ) within the constraints of the system, there is a net effect of the microdroplet expanding until the water activities of the two components become equal. Conversely, when the water activity of the second aqueous medium is higher than the water activity of the first aqueous medium, the microdroplet tends to contract until the water activities become equal. In a useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same, so that any tendency of the microdroplet to contract or expand is continuously offset. Thus, the size of the microdroplet can always be preserved. It was also confirmed that by means of the above, for example, by using the second droplet to supply a cell-growth component at one or more points of any device using