MICRODROPLET MANIPULATION METHOD

Abstract

A method of manipulating microdroplets having an average volume in the range 0.5 femtolitres to 10 nanolitres comprised of at least one biological component and a first aqueous medium having a water activity of a.sub.w1 of less than 1 is provided. It is characterised by the step of maintaining the microdroplets in a water-immiscible carrier fluid which further includes secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1. The method may be employed for example with microdroplets containing biological cells or with microdroplets containing single nucleoside phosphate such as are prepared in a droplet-based nucleic acid sequencer. The method is suitable

Claims

1-17. (canceled)

18. A method of controlling the size of microdroplets and/or maintaining or optimising enzymatic or chemical reactions occurring therein, said microdroplets having an average volume in the range from 0.5 femtolitres to 10 nanolitres comprised of at least one biological component and a first aqueous medium having a water activity of a.sub.w1 of less than 1 comprising the step of maintaining the microdroplets in a water-immiscible carrier fluid which further includes secondary droplets comprised of a second aqueous medium having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.

19. The method of claim 18 for manipulating the size and/or chemical or enzymatic reactivity of the contents of microdroplets having an average volume in the range from 0.5 femtolitres to 10 nanolitres, the microdroplets being comprised of at least one biological component and a first aqueous medium free of biological cells having a water activity of a.sub.w1 of less than 1 wherein the step of maintaining the microdroplets in a water-immiscible carrier fluid further includes secondary droplets comprised of a second aqueous medium and having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 0.5 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.

20. The method of claim 18 for controlling chemical or enzymatic reactivity and/or microdroplet size in microdroplets having an average volume in the range from 4 femtolitres to 10 nanolitres, the microdroplets being comprised of at least one biological cell and a first aqueous medium having a water activity of a.sub.w1 of less than 1 wherein the step of maintaining the microdroplets in a water-immiscible carrier fluid further includes secondary droplets comprised of a second aqueous medium and having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.

21. The method of claim 18, wherein the secondary droplets have a water activity a.sub.w2 which is greater than a.sub.w1.

22. The method of claim 18, wherein the secondary droplets have a water activity a.sub.w2 which is less than a.sub.w1.

23. The method of claim 18, wherein water activities a.sub.w1 and a.sub.w2 are the same.

24. The method of claim 18, wherein a.sub.w1 and a.sub.w2 are independently in the range 0.9 to 1.

25. The method of claim 21, wherein the ionic strength of the second aqueous medium is in the range from 1 to 5 times that of the first aqueous medium.

26. The method of claim 22, wherein the ionic strength of the first aqueous medium is in the range from 1 to 5 times that of the second aqueous medium.

27. The method of claim 18, wherein the average volume of the secondary droplets is less than 10% of the average volume of the microdroplets.

28. The method of claim 18, wherein at least one of the first and second aqueous media further comprise glycerol.

29. The method of claim 18, wherein the biological component is selected from a single nucleoside triphosphate derived from a target nucleic acid, an oligonucleotide derived from the DNA or RNA of a cell, an enzyme or a cell.

30. The method of claim 29, wherein the first and/or second aqueous media are buffers.

31. A method of causing the cellular proliferation of one or more cell types contained within a microdroplet having an average volume in the range 4 femtolitres to 10 nanolitres and comprised of an aqueous buffer comprising the steps of incubating the cell(s) inside the droplets in suitable environmental conditions and thereafter detecting the number of cells inside each droplet, wherein the microdroplets are suspended in an immiscible carrier fluid further comprising secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.

32. A method of analysing or detecting one or more phenotypic traits, genetic traits or protein expression profiles of a cell under consideration, that cell being contained within a microdroplet having an average volume in the range 4 femtolitres to 10 nanolitres and comprised of an aqueous buffer comprising the steps of labelling a target derived from the cell(s) wherein the microdroplets are suspended in an immiscible carrier fluid further comprising secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.

33. A method of sequencing comprising the steps of progressively digesting by pyrophosphorolysis a nucleic acid analyte into an ordered stream of nucleoside triphosphate molecules and generating therefrom a corresponding ordered stream of microdroplets having an average volume in the range from 0.5 femtolitres to 10 nanolitres and each comprised of one of the nucleoside triphosphate molecules and aqueous buffer; reacting each nucleoside triphosphate molecule within each microdroplet with a nucleobase-specific fluorescent probe and thereafter detecting the corresponding fluorescence associated with each microdroplet thereby identifying the nucleobase wherein the microdroplets are suspended in an immiscible carrier fluid further comprising secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 0.5 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.

34. The method of claim 31, wherein the ratio of the water activities of the microdroplets and the secondary droplets is in the range 0.9:1 to 1:0.9; preferably 0.95:1 to 1:0.95.

Description