Microdroplet manipulation method

Abstract

To provide a method of manipulating microdroplets which have an average volume within the range of 0.5 femtoliter to 10 nanoliters and comprise at least one biological component and a first aqueous medium having a water activity aw1 of less than 1.SOLUTION: A method of manipulating microdroplets is characterized by a step of maintaining the microdroplets in a water-immiscible carrier fluid which further includes secondary droplets having an average volume less than 25% of an average volume of the microdroplets up to and including a maximum of 4 femtoliters and wherein a volume ratio of carrier fluid to a total volume of microdroplets per unit volume of the total is greater than 2:1. The method may be used 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 for controlling for example cellular, chemical or enzymatic processes and / or microdroplet size in microdroplets or single nucleotide nucleic acid sequencing.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an improved method for manipulating aqueous microdroplets optionally containing biological cells in an immiscible carrier liquid such as oil. It enables controlling or adjusting the size of the microdroplets and maintaining or optimizing any enzymatic or chemical reactions occurring therein for a predetermined period of time. It enables controlling or adjusting the size of the microdroplets and maintaining or optimizing any enzymatic or chemical reactions occurring therein for a predetermined period of time. It enables controlling or adjusting the size of the microdroplets and maintaining or optimizing any enzymatic or chemical reactions occurring therein for a predetermined period of time. It enables controlling or adjusting the size of the microdroplets and maintaining or optimizing any enzymatic or chemical reactions occurring therein for a predetermined period of time.

Background Art

[0002] In our previous patent applications, such as International Publication No. 2014 / 167323, International Publication No. 2 015 / 121675, International Publication No. 2016 / 012789, International Publication No. 2017 / 140839, and PCT / EP2018 / 066574, a series of analyses including DNA and RNA sequencing, and detection and characterization of cells and viruses were carried out with the aim of manipulating biological components such as cells, enzymes, oligonucleotides, and even single nucleotides within microdroplets. In some embodiments, these methods involve using electro-wetting propulsion forces or directly printing microdroplets onto a substrate coated with a carrier liquid to move the microdroplets dispersed in an immiscible carrier liquid along a microfluidic path within an analytical device. In many examples 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 of the enzymatic processes occurring inside. We have found that this can sometimes interfere with some or all of the enzymatic processes occurring inside. In other examples, it may be desirable to intentionally shrink or expand the size of the microdroplets in a part of the device when a given analysis is being performed. In other examples, it may be desirable to intentionally shrink or expand the size of the microdroplets in a part of the device when a given analysis is being performed. In other examples, it may be desirable to intentionally shrink or expand the size of the microdroplets in a part of the device when a given analysis is being performed.

Summary of the Invention

[0003] In this study, we developed a method for manipulating microdroplets to overcome these problems. This method may 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 present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. w1 For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. For example, it may be used to manipulate the size and / or reactivity of the contents of the microdroplets or to control chemical or enzymatic reactions occurring therein. The present invention is as defined in the appended claims. According to a first aspect of the present invention, there is provided an inclusive method for manipulating microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the method comprising a first aqueous medium having at least one biological component and a water activity a less than 1, the method comprising controlling the size and / or chemical composition of the contents of the microdroplets, the method comprising maintaining the microdroplets in a water-immiscible carrier liquid further comprising secondary droplets of a second aqueous medium having an average volume of less than 25% of the average volume of the microdroplets and up to 4 femtoliters, the method being characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is greater than 2:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0004]

Figure 1

Figure 2

[0005] While not wishing to limit the scope of the present invention, the present invention interacts with the microdroplets without adversely affecting the overall properties of the microdroplets or the effectiveness of any detection method applied to the microdroplets. It is contemplated that the problem can be solved by using a carrier liquid containing very small secondary droplets that can interact with the microdroplets without adversely affecting the overall properties of the microdroplets or the effectiveness of any detection method applied to the microdroplets. When the carrier medium is oil, such a composite medium may be referred to as "hydrated oil". An important feature in this regard is that the relative water activities of the microdroplets and the secondary droplets are controlled within certain parameters and, optionally, are controlled by continuous monitoring and / or feedback loops. Here, the water activity (a ) of an aqueous medium 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 from high water activity to low water activity, within the constraints of our system, when the water activity (a ) of a second aqueous medium is higher than the water activity of a first aqueous medium (a w ), we have found that the net effect is that the microdroplets expand until the water activities of the two components are equal. Conversely, when the water activity of the second aqueous medium is higher than the water activity of the first aqueous medium, the microdroplets tend to shrink until these water activities are equal. In one useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same such that any tendency for the microdroplets to shrink or expand is continuously canceled out. Thus, the size of the microdroplets may be constantly maintained. Also, by using these secondary droplets, for example, the secondary droplets are used to supply cell growth components to the microdroplets at one or more points of any device employing this method. (a w2 ), the microdroplets expand until the water activities of the two components are equal. Conversely, when the water activity of the second aqueous medium is higher than the water activity of the first aqueous medium, the microdroplets tend to shrink until these water activities are equal. (a w1 ), we have found that the net effect is that the microdroplets expand until the water activities of the two components are equal. Conversely, when the water activity of the second aqueous medium is higher than the water activity of the first aqueous medium, the microdroplets tend to shrink until these water activities are equal. In one useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same such that any tendency for the microdroplets to shrink or expand is continuously canceled out. Thus, the size of the microdroplets may be constantly maintained. In one useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same such that any tendency for the microdroplets to shrink or expand is continuously canceled out. Thus, the size of the microdroplets may be constantly maintained. In one useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same such that any tendency for the microdroplets to shrink or expand is continuously canceled out. Thus, the size of the microdroplets may be constantly maintained. In one useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same such that any tendency for the microdroplets to shrink or expand is continuously canceled out. Thus, the size of the microdroplets may be constantly maintained. In one useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same such that any tendency for the microdroplets to shrink or expand is continuously canceled out. Thus, the size of the microdroplets may be constantly maintained. In one useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same such that any tendency for the microdroplets to shrink or expand is continuously canceled out. Thus, the size of the microdroplets may be constantly maintained. In one useful embodiment, the water activities of the first and second aqueous media may be the same or substantially the same such that any tendency for the microdroplets to shrink or expand is continuously canceled out. Thus, the size of the microdroplets may be constantly maintained. By this means, we have found that it is possible to assist in preserving or even enhancing any enzymatic or chemical reactions occurring in the microdroplets. The first and second aqueous media may have compositions that are the same in one embodiment.

[0006] Thus, in one embodiment of the present invention, the water activities of the first and second aqueous media are, independently, in the range of 0.9 to 1. In another embodiment, the water activity of the first aqueous medium is 0.9 or more and less than 1. In yet another embodiment, the ratio (a :a w1 :a w2 ) of the water activities of the first and second aqueous media is in the range of 0.9:1 to 1:0.9.

[0007] One convenient way to perform the operation is to use the first and second media, which are buffer solutions, and, if necessary, by changing the two relative compositions. For example, in one application, the ionic strength of the first aqueous medium is in the range of 1 to 5 times that of the second aqueous medium, preferably 3 to 5 times. In another application, the ionic strength of the second aqueous medium is in the range of 1 to 5 times that of the first aqueous medium, preferably 3 to 5 times. In yet another application the ionic strength is the same or substantially the same, and the ratio of the ionic strengths is in the range of 3:1 to 1:3 . In one particularly useful embodiment, either or both of the first and second aqueous media may contain glycerol as a component, for example, at different concentrations. In another embodiment the pH of the first and second aqueous media is the same or similar and is within the range of 6.5 to 8 .

[0008] Regarding the secondary droplets, they have an average volume that is much smaller than the average value of the microdroplets , at the limit, are emulsified in the carrier liquid and are femtolitre-sized droplets of a second aqueous medium stabilized by a sheath of molecules of a compatible surfactant such as a non-ionic surfactant or may consist of micelles. In one embodiment, the size of these secondary droplets is less than 10% of the volume of the microdroplets used, preferably less than 5%. In another embodiment, the average volume of the secondary droplets is in the range of 10 to 1% of the average volume of the microdroplets. Suitably, the secondary droplets form part of a stable emulsion in a carrier liquid which is, in one embodiment, an immiscible oil. Suitably, the carrier liquid is selected from mineral oil, silicone oil, or fluorocarbon oil. The oil may further contain surfactants and stabilizers as required. Suitably, the ratio of the volume of the carrier liquid to the total volume of the microdroplets is greater than 3:1, preferably 5:1 or more. The method of the present invention is useful in several applications where biological cells are being analyzed. One example is when culturing immortalized mammalian cells in microdroplets for the purpose of screening individual clonal colonies of cells for desirable characteristics such as protein expression or specific genetic traits. Thus, in a second aspect of the present invention, in one embodiment, a method of causing the proliferation of one or more cell types contained within microdroplets having an average volume in the range from 4 femtolitres to 10 nanolitres and containing an aqueous buffer, comprising incubating the cells within the droplets under suitable environmental conditions and then detecting the number of cells within each droplet,

[0009] wherein the microdroplets are suspended in an immiscible carrier liquid having an average volume of less than 25% of the average volume of the microdroplets and further containing secondary droplets up to 4 femtolitres. ​​​​​​​ Characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is 2: 1 and greater methods are provided.

[0010] In another embodiment, a method for detecting one or more phenotypic traits, genetic traits, or protein expression profiles of cells under consideration, wherein the cells have an average volume in the range of 4 femtoliters to 10 nanoliters and are contained within microdroplets containing an aqueous growth medium, and the target derived from the cells is labeled with a fluorescent probe and then the output from the probe is detected, wherein the cell-containing microdroplets have an average volume of less than 25% of the average volume of the microdroplets and are further suspended in an immiscible carrier liquid containing secondary droplets up to a maximum of 4 femtoliters, characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is Greater than 2:1 methods are also provided. Suitable fluorescent probe molecules for this purpose are well known and include fluorescently labeled antibodies, FRET reporter probes that are degraded in the presence of the target protein, and enzyme-labeled antigens.

[0011] In another embodiment, it is a method for analyzing oligonucleotides derived from biological cells having an average volume in the range of 4 femtoliters to 10 nanoliters and further contained within microdroplets containing an aqueous buffer, wherein the oligonucleotides are labeled with a fluorescent hybridization probe and then the corresponding fluorescence is detected, and the microdroplets have an average volume of less than 25% of the average volume of the microdroplets and further contain secondary droplets up to a maximum of 4 femtoliters and are suspended in an immiscible carrier liquid, characterized in that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is such that the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is A method is provided in which the ratio of the volume of the carrier liquid to the total volume of the droplets is greater than 2:1.

[0012] Fluorescent hybridization probes that can be used for this purpose are well known in the art and include molecular beacons, TaqMan® probes, Scorpion® probes, and LNA® probes. Methods for detecting the fluorescence that occurs in all of these embodiments are well known to those skilled in the art and, for example, an incident electromagnetic radiation source (laser, LED, etc.) and a corresponding photodetector for detecting fluorescent photons are used, and there is a method of outputting a data stream that can be analyzed using a microprocessor algorithm. Thus, the target in these methods may be the cell itself, one or more oligonucleotides derived therefrom, or a product such as a protein expressed by the cell when cultured in the microdroplet itself. Such oligonucleotides may be generated from the cell by lysis.

[0013] The method of the present invention may also be suitably used in connection with biological components that are acellular or cell-free, but in one embodiment may be used to manipulate nucleic acids or components thereof that were previously derived from biological cells. Thus, in a third aspect of the present invention, a method of manipulating the contents and / or reactivity of microdroplets having an average volume in the range of 0.5 femtoliters to 10 nanoliters, the microdroplets comprising at least one biological component and a first aqueous medium that does not contain biological cells having a water activity a less than 1.

[0014] w1 , maintaining the microdroplets in a water-immiscible carrier liquid containing a second aqueous phase and having an average volume of less than 25% of the average volume of the microdroplets, further comprising secondary droplets up to a maximum of 0.5 femtoliters, characterized by a step, and the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is provided, which is greater than 2:1.

[0015] The method of the third aspect of the present invention is useful for many applications where the biological component is a single nucleotide, for example, a single nucleoside triphosphate or a single nucleoside monophosphate. For example, this method can be advantageously used together with one of the sequencing methods we previously described, for example, but not limited to these, such as those described in European Patent Application Publication No. 3013987 or other of the above-mentioned patent applications directed to the reader. Therefore, in the third aspect a nucleic acid analyte is first digested by adding pyrophosphorolysis to an ordered stream of nucleoside triphosphate molecules, from which an average volume in the range of 0.5 femtoliters to 10 nanoliters is obtained, generating a corresponding ordered stream of microdroplets each containing one of the nucleoside triphosphate molecules and an aqueous buffer, reacting each nucleoside triphosphate molecule in each microdroplet with a fluorescent probe specific for the nucleic acid base, and then detecting the corresponding fluorescence associated with each microdroplet, thereby identifying the nucleic acid base, characterized in that the microdroplets are suspended in an immiscible carrier liquid having an average volume of less than 25% of the average volume of the microdroplets and further containing secondary droplets up to a maximum of 0.5 femtoliters, and the ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume of the whole is provided, which is greater than 2:1.

[0016] Fluorescent probes suitable for use in this application are described in our previous patent applications, such as International Publication No. 2016 / 012789 and subsequent published applications directed to the reader. These probes are (a) non-fluorescent in the unused state, and (b ) capable of undergoing exonuclease digestion once used to generate a detectable fluorophore bound to a single nucleotide monophosphate. The resulting fluorescence can be detected and analyzed as described above. In all these further aspects of the invention, the ratio of the water activity of the first and second aqueous media associated with the microdroplets and the secondary droplets respectively is in the range of 0.9:1 to 1:0.9, preferably 0 .95:1 to 1:0.95, and for example, preferably 1:1.

[0017] In all these further aspects of the invention, the ratio of the water activity of the first and second aqueous media associated with the microdroplets and the secondary droplets respectively is in the range of 0.9:1 to 1:0.9, preferably 0 .95:1 to 1:0.95, and for example, preferably 1:1.

Examples

[0018] The advantageous effect of hydrating the carrier phase as described above is shown by the following examples.

[0019] Example 1 (Cell proliferation) A continuous oil phase material is prepared by mixing 99 parts of a hydrogen fluoride ether continuous phase and 1 part of a fluorinated surfactant. An equal volume of the oil / surfactant mixture and a small amount of RPMI 1640 medium (Thermo Fisher Scientific, UK) are mixed, and the mixture is stirred at 37 ° C for 24 hours to form a polydisperse emulsion to prepare a growth medium-treated carrier phase. Then, this emulsion is allowed to stand until it spontaneously fractionates to form an upper phase containing large droplets of aqueous growth medium and undispersed plugs, and a lower phase containing only the smallest vesicles of growth medium suspended in an oil phase that is now further saturated with the dissolved aqueous medium . ​​​​​ This subphase is removed from the container using a pipette and stored for later use.

[0020] Jurkat E6-1 T cell lymphoma cells (ATCC, Virginia, USA) are suspended in RPMI medium at a concentration of 8E6 cells / mL. Next, this medium and cells are flowed through an emulsifying device to form droplets with a diameter of 50 μm and the cells are dispersed throughout the droplets. The outer carrier phase of the emulsion is hydrogen fluoride ether oil mixed with 1% of a suitable surfactant to stabilize the droplets in the solution. The emulsion thus formed spontaneously fractionates to form a layer of densely packed monodisperse aqueous droplets that float on top of a column of continuous oil / surfactant mixture. Next, the emulsion is gently mixed to disperse it uniformly and divided into three aliquots containing the droplets and the carrier phase. One aliquot (initial reference) is immediately transferred to a hemocytometer flow cell and the droplets therein are examined using a 20x magnifying optical microscope. The cell occupancy of each droplet is recorded by counting the number of separate cells within each droplet. Empty droplets are ignored. The second aliquot is fractionated again and the lower carrier phase is removed using a pipette.

[0021] To replace the removed untreated carrier phase, an equal volume of previously treated carrier phase is introduced into the sample. The third aliquot is left unchanged. Next, both the second and third aliquots are transferred to a partially sealed container that allows gas permeation between the container and its surroundings. Both containers are placed in an environmental control CO2 incubator set to contain a 5% CO2 / air mixture, 95% humidity, and a set temperature of 37°C. The aliquots are incubated for 24 hours

[0022] The second aliquot is fractionated again and the lower carrier phase is removed using a pipette. To replace the removed untreated carrier phase, an equal volume of previously treated carrier phase is introduced into the sample. The third aliquot is left unchanged. Next, both the second and third aliquots are transferred to a partially sealed container that allows gas permeation between the container and its surroundings. Both containers are placed in an environmental control CO2 incubator set to contain a 5% CO2 / air mixture, 95% humidity, and a set temperature of 37°C. The aliquots are incubated for 24 hours Both containers are placed in an environmental control CO2 incubator set to contain a 5% CO2 / air mixture, 95% humidity, and a set temperature of 37°C. The aliquots are incubated for 24 hours Both containers are placed in an environmental control CO2 incubator set to contain a 5% CO2 / air mixture, 95% humidity, and a set temperature of 37°C. The aliquots are incubated for 24 hours Incubate.

[0023] Next, remove these aliquots from the incubator and introduce them onto a hemocytometer for inspection and analysis in the same manner as the reference aliquots. Then, the change in the cell population distribution after incubation (characteristics of cell growth) can be compared between different oil treatments.

[0024] Figure 1 compares the results obtained after 24-hour culture with the baseline measurements at 0 hours and 24 hours without hydration of the oil. Here, cell growth is represented as the fraction of droplets containing multiple cells. It will be seen that the oil is improved when hydrated in the cell culture medium as compared to the baseline cell growth.

[0025] Example 2 (reactivity) Prepare a continuous hydrated oil phase by mixing 99 parts by weight of light oil and 1 part by weight of a pegylated surfactant. To completely mix the oil and the surfactant, leave the oil in a rotator overnight. Prepare a hydrated oil by mixing 5 parts of oil and 3 parts of an aqueous hydration phase. The aqueous hydration phase consists of either the same physiological saline buffer used in the dispersed emulsion phase or water only. Rotate the mixture at 50 °C overnight and then at 70 °C for 60 minutes. Let the emulsion stand for 15 minutes. Aliquot the upper part of the emulsion and then centrifuge the aliquot to adjust the hydration level of the oil. The longer the centrifugation time, the lower the hydration level. Measure the hydration level using a Karl Fischer titrator. Once the appropriate hydration level (usually 500 - 1000 ppm) is reached, pipette the supernatant of the aliquot into a new tube and freeze it until use.

[0026] ​​​​​​​ Mix 8 volume parts of oil (hydrated or non-hydrated) with 1 volume part of dispersed aqueous phase, followed by vortexing Mix for 5 minutes with a magnetic stirrer and centrifuge for 1 minute at 400 RPM to produce a multi-droplet emulsion. Pipette the upper half of the mixture into a new tube and centrifuge for 5 seconds Transfer the emulsion from the bottom of the tube with a pipette for further use .

[0027] For the measurement of enzyme activity, the above-mentioned dispersed aqueous phase consists of single nucleotide detection chemistry as already described and exemplified in European Patent Application Publication No. 3013987 book.

[0028] For the measurement of fluorescence intensity, sandwich the emulsion between two transparent substrates separated by a spacer corresponding to the average emulsion droplet size. When excited with light in an appropriate wavelength range the fluorescence signal emitted from each emulsion droplet is measured along with the diameter of the droplet collected from the bright field image of the emulsion .

[0029] The data shown as a histogram in Figure 2 shows the average fluorescence intensity of 6 μm droplets incubated in non-hydrated oil ("dry oil") in oil hydrated with water ("water only") or in oil hydrated with 3 times the buffer concentration of the droplets ("3 × buffer"). Droplets incubated in non-hydrated oil show very low intensities above the background of about 1000 counts in these samples. Droplets incubated in oil hydrated with water show an increase in intensity compared to droplets incubated in non-hydrated oil. Incubating the droplets in oil hydrated with 3 times the buffer concentration results in a further increase in the average intensity . ​​It can be seen that it increases. This indicates that the hydration of both oils can be used to maintain the enzymatic reactivity in these droplets.

[0030] Example 3 (Droplet size effect) Microdroplets are deposited on a substrate immersed in a continuous oil phase, as already described, for example, in European Patent Application Publication No. 3008207 directed to the reader.

[0031] A continuous hydrated oil phase is prepared by mixing 99 parts by weight of paraffin oil and 1 part by weight of a pegylated surfactant. To completely mix the oil and the surfactant, the oil is left in a rotator overnight. Hydrated oil is prepared by mixing 5 parts of oil and 3 parts of an aqueous hydrated phase. The aqueous hydrated phase consists of either water containing 4% glycerol or water without it. The mixture is rotated at 50 °C overnight and then at 70 °C for 60 minutes. The emulsion is left standing for 15 minutes. The upper part of the emulsion is dispensed, and then the aliquot is centrifuged to adjust the hydration level of the oil. The longer the centrifugation time, the lower the hydration level. The hydration level is measured using a Karl Fischer titrator. Once the appropriate hydration level (usually 500 - 1000 ppm) is reached, the supernatant of the aliquot is transferred to a new tube with a pipette and frozen until use.

[0032] The dispersed aqueous phase consists of water containing 4% glycerol or water without it. The deposited droplets are subjected to an incubation cycle at 70 °C for 115 minutes. Next, the diameter of the droplets of the emulsion is measured from the bright-field microscope image and compared with the diameter measured before the incubation cycle to infer the shrinkage or growth of the droplets.

[0033] ​​​​​​​​​​​​​​The data shown below represents the change in the average volume of droplets during the high-temperature incubation step, as a function of the percentage of glycerol in the oil hydration and in the droplets, respectively. In the reference sample, when glycerol is not present in either the oil hydration or the droplets, the droplets shrink on average. When glycerol is present in the droplets but not in the oil hydration, the addition of glycerol to the droplets causes the water activity in the oil to be higher than that in the droplets, so the droplets grow compared to the reference. When glycerol is added to the oil hydration but not to the droplets, the opposite occurs. Since the water activity of the droplets is higher than that of the oil, the droplets shrink compared to the reference. This shows that the shrinkage and growth of droplets can be controlled using specific contents of droplets and oil hydration.

[0034] [Table 1]

Claims

1. At least one biological component and a water activity a of less than 1. ｗ１ A first aqueous medium having and a microfluidic device having an average volume in the range of 0.5 femtoliters to 10 nanoliters. A method of manipulating droplets, comprising: dissolving the microdroplets in a second aqueous medium; and further comprising secondary droplets having an average volume of less than 25% of the volume and up to 4 femtoliters. The method is characterized by the step of maintaining the micro-liquid crystals in a water-immiscible carrier liquid containing the micro-liquid crystals, and the micro-liquid crystals per total unit volume are maintained in a water-immiscible carrier liquid containing the micro-liquid crystals. A method wherein the ratio of the volume of the carrier liquid to the total volume of the drops is greater than 2:

1.

2. Among the microdroplets having an average volume ranging from 0.5 femtoliters to 10 nanoliters A method for manipulating the size and / or chemical or enzymatic reactivity of a container, comprising: The droplets comprise at least one biological component and a water activity a of less than 1. ｗ１ Biological cells with a first aqueous medium free of cells, and distributing the microdroplets to a second aqueous medium free of cells, Bidroplets having an average volume less than 25% of the average volume of the small droplets, up to a maximum of 0.5 femtoliters The method further comprises the step of maintaining the droplets in a water-immiscible carrier liquid, and forming an entire unit.

10. The ratio of the volume of the carrier liquid to the total volume of the microdroplets per volume is greater than 2:

1. The method according to

3. In microdroplets having an average volume ranging from 4 femtoliters to 10 nanoliters A method for controlling chemical or enzymatic reactivity and / or microdroplet size, comprising: The droplets contain at least one biological cell and a water activity a of less than 1. ｗ１ A first water having and a second aqueous medium, the microdroplets being dispersed in a second aqueous medium that is 25% of the average volume of the microdroplets. and further comprising secondary droplets up to 4 femtoliters. wherein the total volume of the microdroplets per unit volume of the total volume of the carrier liquid is 2. The method of claim 1 , wherein the ratio of the volume of carrier liquid to the volume of carrier liquid is greater than 2:

1.

4. The secondary droplets are ｗ１ Water activity a greater than ｗ２ Claim 1 4. The method according to any one of 1 to 3.

5. The secondary droplets are ｗ１ Water activity a smaller than ｗ２ Claim 1 4. The method according to any one of 1 to 3.

6. The water activity a ｗ１ and a ｗ２ Any one of claims 1 to 3, characterized in that 3. The method according to claim 1 .

7. a ｗ１ and a ｗ２ are independently in the range of 0.9 to 1, 2. The method according to claim 1 .

8. 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.

5. The method of claim 4, wherein the

9. The ionic strength of the first medium is in the range of 1 to 5 times the ionic strength of the second aqueous medium. The method according to claim 5, characterized in that

10. The average volume of the secondary droplets is less than 10% of the average volume of the microdroplets. The method according to any one of claims 1 to 9,

11. At least one of the first and second aqueous media further comprises glycerol. The method according to any one of claims 1 to 10, characterized in that

12. The biological component may be a single nucleoside triphosphate derived from a target nucleic acid, a DNA fragment of a cell, or or an oligonucleotide derived from RNA, an enzyme, or a cell. The method according to any one of claims 1 to 11,

13. 13. The method according to claim 12, characterized in that the first and / or second aqueous medium is a buffer solution. method.

14. having an average volume ranging from 4 femtoliters to 10 nanoliters and containing an aqueous buffer solution A method for inducing cell proliferation of one or more cell types contained within a microdroplet, the method comprising: Incubate the cells in the droplets under appropriate environmental conditions and then detect the number of cells in each droplet. wherein the microdroplets have an average volume that is less than 25% of the average volume of the microdroplets. and further comprising up to 4 femtoliters of secondary droplets suspended in an immiscible carrier liquid. The volume of the carrier liquid relative to the total volume of the microdroplets per unit volume of the whole is The ratio of is greater than 2:

1.

15. One or more phenotypic traits, genotypic traits, or protein expression profiles of the cells under study. The method of analyzing or detecting cells, wherein the cells are in a volume of between 4 femtoliters and 10 nanoliters. and the cells are contained within microdroplets comprising an aqueous buffer, the microdroplets having an average volume in the range of 1000 to 10000 μg / ml. labeling the target, wherein the microdroplet is less than 25% of the average volume of the microdroplet; and further comprising secondary droplets up to 4 femtoliters. The droplets are suspended in a liquid, and the ratio of the total volume of the droplets to the total unit volume is The ratio of the volume of carrier liquid to the volume of carrier liquid is greater than 2:

1.

16. A method for sequencing in which a nucleic acid analyte is added to an ordered stream of nucleoside triphosphate molecules. It is gradually digested by phosphorolysis, and from there, 0.5 femtoliters to 10 nanoliters and each of said nucleoside triphosphate molecules and an aqueous buffer solution is mixed with said nucleoside triphosphate molecule. generating a corresponding ordered stream of microdroplets each containing a nucleus; and The oligosaccharide triphosphate molecule is reacted with a fluorescent probe specific to the nucleic acid base, and then each microfluidic and detecting corresponding fluorescence associated with the droplets, thereby identifying said nucleobases. and the microdroplets have an average volume that is less than 25% of the average volume of the microdroplets and is up to 0. The secondary droplets are suspended in an immiscible carrier liquid containing up to 5 femtoliters of the secondary droplets. The ratio of the volume of the carrier liquid to the total volume of the microdroplets per unit volume is A method greater than 2:

1.

17. The ratio of water activity of the microdroplets to the secondary droplets is 0.9:1 to 1:0.9, preferably 0 17. Any of claims 14 to 16, characterized in that the ratio is in the range of .95:1 to 1:0.

95.

13. The method according to claim 1.

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