GB2495182A

GB2495182A - System and Method for splitting droplets in a plug flow

Info

Publication number: GB2495182A
Application number: GB1215443.1A
Authority: GB
Inventors: Tomasz Kaminski; Piotr Garstecki
Original assignee: Instytut Chemii Fizycznej of PAN
Current assignee: Instytut Chemii Fizycznej of PAN
Priority date: 2011-09-30
Filing date: 2012-08-30
Publication date: 2013-04-03
Anticipated expiration: 2032-08-30
Also published as: GB2495182B; PL396493A1; PL229042B1; GB201215443D0

Abstract

A method for splitting droplets suspended in a carrier liquid in a microfluidic device comprising supplying the droplet 1 to be split to a junction 7 in a channel 2, 4, 5 and breaking up the droplet at the junction 7 into smaller droplets 8 using a stream of cutting fluid 6 which is immiscible with the droplet, characterised in that the channel before the junction has a narrowed section 4, 5 wherein the length of the narrowed section is at least equal to the longest dimension of the cross section of the channel 2 before the narrowing, preferably at least equal to a hundredfold of that dimension, and most preferably at least equal a thousandfold of that dimension, and the longest dimension of the cross section of the narrowed section of the channel 4, 5 is from 1 to 5 times less than the longest dimension of the cross section of the microfluidic channel 2 before the narrowing, preferably from 2 to 3 times less than that dimension. The method causes the droplet to be elongated before reaching the junction. At the junction, the droplet is split by perpendicular streams of immiscible liquid. The emerging droplets are monodisperse and have a decreased volume. A system for performing the method is also disclosed.

Description

Method for splitting droplets in microfluidic junction and system for splitting droplets in microfluidic junction The subject matter of the invention isa method for splitting droplets in a microfluidic junction, comprising the stage of supplying droplets to the microfluidic junction and droplet breakup in the junction. The invention comprises also a system for droplet splitting in the microfluidic junction that allows making use of the method. The invention finds application in microfluidics.

The solutions according to the present invention, in combination with microfluidic modules known in the state of the art -for instance those described in earlier patent applications of Prof. Piotr Garstecki's research team (Polish patent applications no. P- 390250, P-390251, P-393619 not published yet, and an international patent application no. PCTJPL2O11/050002) may be used to perform high-throughput screening tests carried out in droplets of picoliter (subnanoliter) volumes that can accommodate single cells or single molecules. Reactions on single cells have a huge potential in diagnostics and in studies on heterogeneous populations including, e.g., cancer tissues or environmental microbiological samples. In recent years, the experiments performed on single cells have become the basics in such fields as systems biology, genomics or metabolomics. Furthermore, picoliter droplets produced in our systems can be used as a platform for reactions on single biological molecules including enzymes or nucleic acid sequences. Such reactions are a basis for guided enzyme evolution or digital PCR (Polimerase Chain Reaction -e.g., US RE41,780E).

A growing number of reports on applications of microfluidic systems in biological sciences allow one to predict a rapid development of the lab-on-a-chip technology in near future. Particularly promising is the application of droplets generated in microchannels as miniaturised reactors, because of their small volume, from microliters, through nanoliters down to picoliters.

Typically, droplet-based microsystems possess a multitude of microfluidic channels, with their inlets and outlets that can join inside the system, where droplets of solutions are surrounded by continuous phase immiscible therewith. Further, the droplets inside the 1.

systems can be merged, transported along the channels while their contents are being mixed, stored under specific or varying conditions, and finally sorted or split in channel junctions and recovered from the system. The use of microlaboratories to perform chemical and biochemical reactions inside microdroplets offers the following advantages [F-I. Song, D. L. Chen and R. F. lsmagilov, Any. Chem. mt. Ed., 2006, 45, 7336-7356]: i) no dispersion of time of residence for fluid elements in a channel, ii) rapid mixing, Hi) reaction kinetics can be easily controlled, iv) multiple reactions can be performed in parallel, v) low consumption of reagents, and vi) fast detection of the outcome of reactions (due to a low droplet volume the reaction products reach faster measurable concentrations).

These characteristics make the microdroplet-based microsystems a valuable tool for analytical chemistry, synthetic chemistry, biochemistry, microbiology, medical diagnostics or molecular diagnostics.

One of the challenges related to droplet-based microsystems is formation of droplets with known and varying composition. It would allow performing high-throughput screening tests. Varying droplet composition shall mean here the concentration of active chemical compounds, such as e.g., drugs, inhibitors, hormones, cofactors, etc., as well as more complex molecules such as proteins (enzymes, antibodies) nucleic acids, and macromolecules of other sort, in particular paramagnetic molecules. For many reasons, it is desirable that droplet volume is less than 10 nL, because of i) minimal consumption of reagents, ii) maximal throughput, iii) reactions and biochemical tests can be performed on single cells or molecules.

In the state of the art there are a few solutions related to generating droplets with a volume less than 10 nL and with varying composition. For instance, H. Song and R. F. lsmagilov (J. Am. Chem. Soc. 2003 125: 14613-14619) generated a concentration gradient from one to three reagents inside a droplet by manipulating the flow rate in each of three or four channels supplying the aqueous phase. Such a solution has a number of drawbacks: i) the concentrations cannot be varied independently from each other, H) the concentrations are varied continuously, and not stepwise (discretely), iii) the range of concentrations is narrow and not greater than two orders of magnitude, iv) the concentration inside a droplet is neither fully known nor controlled. In another solution, A. B. Theaberge et al. (Analytical Chemistry, 2010, 82, 3449-3453) showed it was possible to integrate chromatographic column with a microfluidic system. Before being closed inside a droplet, a mixture of active chemical compounds is separated, whereas the separation generates gradients spanning several orders of magnitude. As in the earlier case, however, the researcher has a very little control over the droplet composition: i) separation characteristics must be known for each substance and for each type of chromatographic column, H) simultaneous concentration screening is not possible for mixtures of two or more active compounds -for instance to study interactions between them, iii) very many excessive droplets are generated which significantly reduces the throughput of the system. Another solution is to use droplet libraries [patent application US 2010/0022414]. The solution consists in creating populations of monodisperse microdroplets that form a stable emulsion. A population is a mixture of droplets of different compositions -each sub-population of droplets with identical composition is coded by a marker, or by a mixture of markers. The markers are mostly fluorescent compounds, but may be also chemicals containing radioisotopes, enzymes or other markers, e.g., DNA sequences. The reaction consists in a high-throughput merging of droplets from a library with droplets containing cells, enzymes or analyte of another sort. [.

Brouzes et al. [Proceedings of the National Academy of sciences of the United States of America, 2009, 106, 14195-14200] reported that the libraries allow for evaluation of drug cytotoxicity on the level of single mammalian cells. In another example, N. K. ioensson et al. (Angew. Chem. Int. Ed. 2009, 48, 2518 -2521) showed that it is possible to detect rare biomarkers on the surface of single mammalian cells. A drawback of the technology of droplet libraries at the present level is that each sub-population must be generated separately, which is a laborious and costly process. Moreover, production of a library is very difficult while keeping consumption of reagents at the minimum -filling the classical system and stabilisation of the droplet formation process require at least several microliters of reagents (which generates significant costs if fluorescent markers are used).

Alternative methods for producing microdroplets of known volumes are systems allowing for aspiring fluids from external sources, such as, e.g., multi-well titration plates.

For instance, J. Clausell-Tormos (Lab Chip, 2010, 10, 1302-1307) reported an automatic sampling system exploiting a multi-channel valve used in chromatography. The samples of substances were sampled from a multi-well plate, and subsequently placed in tubing as droplets separated from each other by oil. Chen (PNAS, 2008, 105 (44), 16843-16848) developed a system called,,Chemistrode" that allows for aspiring samples directly from cell culture and forming droplets therefrom. Liu (Lab on a Chip, 2009, 9, 2153-2162) modified this system so as to allow for aspiration of samples from small volumes. Sun (Lab Chip, 2010, 10, 2864-2868) reported an automatic system allowing for sampling from test tubes. An important problem in all techniques for depositing or aspiring small fluid samples is to reduce, and preferably to eliminate gas bubbles being introduced into the system. Major limitations of the methods listed above include their slowliness, possible cross-contaminations, and the need to generate earlier the composition of reagents, e.g., using a pipette or an automatic pipetting station.

The only solution allowing for precise control over droplet composition is an automated device reported by K. Churski et al. (Lab Chip, 2010, 10, 816-818.), where droplets on demand with specific volumes are simultaneously produced in several parallel microchannels, and then merged in various proportions to form a large drop with known chemical composition. This drop is, however, of significant volume (>500 nL) that cannot be reduced because of the dead volume of the valves. Due to a so large volume, the microfluidic system cannot be used in numerous applications, such as single-cell tests or single-molecule reactions. Also the results of detection cannot be speeded up, which can be achieved for very small droplets [J. 0. Boedicker et al., Lab Chip, 2008, 8, 1265-1272].

In the state of the art there is still no microfluidic system or method allowing for fast splitting of large drops of known composition into populations of small monodisperse droplets with a volume less than 10 nL each. On the other hand, in the state of the art there are several methods for splitting droplets in a T-junction in a microfluidic system, where the continuous phase (i.e., the phase wetting channel walls) is liquid [ft R. Link, S. L. Anna, D. A. Weitz, and H. A. Stone. Phys. Rev. Lett. 2004, 92, 4.], [J. Nie and R. T. Kennedy, Anal. Chem. 2010, 82, 7852-7856]. Using an appropriate geometry it is possible to break up a droplet in a ratio reversely proportional to the flow resistances in each of the two branches of a 1-junction. An unfavourable characteristic of this solution is the fact that the initial droplet is split into two identical droplets only. In order to split a large drop into a greater number of droplets, the number of T-junctions must be multiplied. This solution is inconvenient in microfabrication and has limited stability of operation. For example, splitting a drop into 1000 smaller droplets requires creating a system composed of 1000 parallel channels, and defective fabrication of, or stopping the flow in one of them perturbs the operation of the whole device. According to J. Clausell-Tormos et al. (Lab Chip, 2010, 10, 1302-1307), splitting a large drop into eight monodisperse droplets is already problematic, and the operation requires additional solutions to stabilise pressures inside channels. Making use of that solution to split drops into a greater number of small droplets seems technologically unfavourable.

According to the invention, the method for splitting droplets in a microfluidic junction comprising the stage of a) supplying the droplet to be split, suspended in a carrier liquid, to a microfluidic junction through a microfluidic channel, and the stage of b) breaking up the droplet to be split in a microfluidic junction into smaller droplets using a stream of cutting fluid, immiscible with the liquid the droplet to be split is made of, characterised in that the said stage a) of supplying the droplet to be split, suspended in a carrier liquid, to the microfluidic junction through a microfluidic channel comprises transporting the droplet to be split through a narrowed section of the microfluidic channel, whereas: i) the length of the narrowed section of the microfluidic channel is at least equal to the longest dimension of the cross section of the microfluidic channel before the narrowing, preferably at least equal to a hundredfold of that dimension, and most preferably at least equal a thousandfold of that dimension, and H) the longest dimension of the cross section of the narrowed section of the microfluidic channel is from 1 to 5 times less than the longest dimension of the cross section of the microfluidic channel before the narrowing, preferably from 2 to 3 times less than that dimension.

Preferably, Hi) the cross section area of the narrowed section of the microfluidic channel is from 1 to 25 times less than the cross section area of the microfluidic channel before the narrowing, more preferably from 4 to 10 times less than the cross section area of the microfluidic channel before the narrowing.

Preferably, iv) the ratio in which the maximal dimension of the cross section of the microfiuidic channel changes as a result of narrowing thereof, to the length of the microfluidic channel on which the change takes place, is not less than 1.15, and not greater than 3.46, and more preferably is equal to 2.

Particularly preferably, the said narrowed section of the microfluidic channel is composed of a series of narrower and narrower subsections, preferably from two to ten subsections, whereas each two consecutive subsections fulfil conditions i) and H) as set forth above, and more preferably also condition iii) and/or condition iv), as set forth above.

Preferably, the said microfluidic junction is a flow-focusing junction, T-junction or any other junction allowing for breaking up droplets into smaller ones.

Preferably, the said microfluidic channels are channels with rectangular, square or circular cross sections.

Preferably, the said droplet to be split has a volume from 100 nL to 100 pL.

In a preferred embodiment of the invention, the droplet to be split is being split into minimum 100, more preferably minimum 1000, yet more preferably minimum 10000, yet more preferably minimum 100000, and most preferably minimum 1000000 smaller droplets.

In a further preferred embodiment of the invention, a sequence of droplets to be split is formed, and the stages a) and b) are repeated for each droplet in such a sequence.

Particularly preferably, the collections of smaller droplets resulting from splitting of each droplet to be split in the said sequence are drained to the drain channel located behind the microfluidic junction, and the collections of smaller droplets are separated from each other by separating droplets produced from a third fluid, immiscible both with the fluid used for producing smaller droplets and with the carrier liquid.

Preferably, the said third fluid is air, or silicone oil, fluorinated oil, hydrocarbon oil, or mineral oil.

Preferably, the ratio of the flow rate of the cutting stream and the flow rate of the droplets to be split is from 1.5:1 to 3:1.

Preferably, the droplets to be split contain surfactant in an amount less than 1% by weight, preferably less than 0.1% by weight, and the cutting stream contains surfactant in an amount over 2% by weight.

The invention comprises also a system for splitting droplets in a microfluidic junction comprising a microfluidic junction and a microfluidic channel for supplying droplets to be split to the microfluidic junction, characterised in that the said microfluidic channel for supplying droplets to be split to the microfluidic junction comprises a narrowed section of the microfluidic channel, whereas: i) the length of the narrowed section of the microfluidic channel (4, 5) is at least equal to the longest dimension of the cross section of the microfluidic channel before the narrowing, preferably at least equal to a hundredfold of that dimension, and most preferably at least equal a thousandfold of that dimension, and ii) the longest dimension of the cross section of the narrowed section of the microfluidic channel is from 1 to 5 times less than the longest dimension of the cross section of the microfluidic channel before the narrowing, preferably from 2 to 3 times less than that dimension.

Preferably, iv) the ratio in which the maximal dimension of the cross section of the microfluidic channel changes as a result of narrowing thereof, to the length of the microfluidic channel on which the change takes place, is not less than 1.15, and not greater than 3.46, and more preferably is equal to 2.

Particularly preferably, the said narrowed section of the microfluidic channel is composed of a series of narrower and narrower subsections, preferably from two to ten subsections, whereas each two consecutive subsections fulfil conditions i) and H) as set forth above, and more preferably also condition Hi) and/or condition iv), as set forth above.

Preferably, the said microfluidic junction is a flow-focusing junction, 1-junction or any other junction allowing for breaking up droplets into smaller ones.

Particularly preferably, additionally, behind the said microfluidic junction, the system has a drain channel and a side channel (27), connecting to the said drain channel in a 1- junction, whereas the said side channel is used to supply the third fluid to the said 1-junction.

The solution presented in this patent application enables splitting of droplets in a microfluidic junction, in particular in a flow-focusing junction, as well as grouping and separation of emerging droplets with a volume less than 10 nL in separate populations, so called droplet libraries". The use of a module to synchronously produce droplets on demand, whereas said droplets are composed of a third fluid, immiscible with both the continuous phase and the droplet phase, provides a physical protection for the populations of small droplets against mutual mixing with each other. It excludes the necessity to use fluorescent markers or any other markers. Alternatively, the device may be used for encoding existing libraries with fluorescent compounds or other chemicals.

The microfluidic device allowing for splitting a large (>100 nL) drop into a population of small droplets with a volume of (<10 ilL) comprises at least one narrowing of a channel and a narrowed channel of appropriate length so as to accommodate the whole large drop.

Furthermore, the system comprises at least one microfluidic junction, preferably a flow- focusing junction wherein the droplet splitting takes place, and -preferably -at least one T-junction, wherein so called spacer droplets are generated to separate the population of small droplets and to prevent from mixing droplets from different sub-populations and differing by chemical composition. These droplets must be made of a fluid that is immiscible both with the droplet phase and the continuous phase. Such a fluid may be a gas (e.g., air, nitrogen or any other gas that is inert for the course of reaction), or a liquid, e.g. silicone oil in a water/perfluorinated oil system.

The Inventors of the present invention were able to split droplets produced inside a microfluidic system into numerous populations of monodisperse droplets with volumes less than 10 nL. In the method according to the invention, synchronisation of droplet splitting, with minimal consumption of the continuous phase, and production of droplets separating "droplet libraries" can be reached either exclusively by setting appropriate shifts between electrical signals triggering droplet production and splitting thereof, or by coupling the control over the valves controlling the flow in the system with signals providing information about droplet position in the system.

Further, it turned out unexpectedly that the system fabricated according to the invention allows for encapsulating single cells, microspheres, and other macroobjects of analytical importance. Populations of droplets (<10 nL), separated by large drops of immiscible fluid may be incubated for a time period controlled from fractions of seconds to hours. Moreover, the system fabricated according to the invention enables performing sequences of measurements (e.g., fluorescent measurements) on a single droplet, or on a selected subgroup of droplets that are present in the system. The sequences of time-shifted measurements allow for determining the kinetics of reaction or processes occurring inside the droplets. Alternatively, electrocoalescence may be applied to droplet populations to restore the initial drop with large volume, to perform subsequently on the droplet complex asymmetric splitting operations, coalescence with other droplets, spectrophotometric measurement, or another operation that for technical reasons can not be performed on droplets with a volume less than 10 [IL.

Preferred embodiments of the invention Preferred embodiments are now explained with reference to the accompanying figures, wherein: Fig. 1 (A, B) shows a picture of two essential fragments of a typical flow-focusing junction used for high-throughput droplet splitting, according to example 1, Fig. 2 shows a picture of another typical flow-focusing junction used for high-throughput droplet splitting, according to example 2, Fig. 3 shows a picture of a flow-focusing junction that is unsuitable for high-throughput droplet splitting (comparative example 3), Fig. 4 shows a plot presenting size distributions of droplets produced in junctions from Fig. 1, 2, and 3, Fig. 5 shows a plot presenting size distribution of droplets produced in junction from Fig. 1, as a function of surfactant concentration, Fig. 6 shows a plot presenting size distributions of droplets produced in junction from Fig. las a function of flow rate of the oil stream cutting the droplet, Fig. 7 shows a schematic diagram of a microsystem according to the invention, used for high-throughput droplet splitting in a flow-focusing microfluidic junction as well as separation of emerging droplets and formation of droplet libraries, Fig. S shows droplet libraries in tubing, Fig. 9 shows a schematic diagram of a typical system used to generate droplet libraries according to the invention, in accordance with example 9, and Fig. 10 shows a schematic diagram of a typical system used to generate droplet libraries according to the invention, in accordance with example 10.

Example 1

In a preferred example of embodiment, a droplet residing in a microchannel is split in a flow-focusing junction after prior elongation of the entire droplet. In the example schematically depicted in Fig. 1A, droplet 1 residing in a wide channel 2 is being introduced into the narrowing 3 and is elongated in a narrow channel 4. Preferably, the entire droplet is elongated in the channel 3 before reaching the flow focusing junction 7 shown in the scheme lB. Preferably, the narrow channel 4 has dimensions similar to the junction 7, while being wide enough to not allow the droplet to be broken up into two or more droplets during elongation. Preferably, the dimensions of the narrow channel 4 are from 2 to 5 times less than the dimensions of the wide channel 1. Preferably, the cross sections of channels 2.

4 are rectangular, square, or circular, whereas the circular cross section is the most preferable one. The narrowed droplet flows subsequently to the next narrowing 5 that is terminated with a flow focusing junction 7. In the junction 7 the droplet is being split by perpendicular streams of immiscible liquid 6 (e.g., paraffin oil), supplied from a single source. It is characteristic that the emerging droplets S are monodisperse (CV<2%), have volumes less than 10 nL, and do not coalescence with each other. The droplets are collected in a widening channel 9.

Example 2

In a preferred example depicted schematically in Fig. 2, a droplet 11 residing in a wide channel 10 is being introduced into the narrowing 12 and split at junction 14 without prior elongation of the entire droplet. Preferably, the cross sections of channels 10, 12 are rectangular, square, or circular, whereas the circular cross section is the most preferable one. In the junction 14 the droplet is being split by perpendicular streams of immiscible liquid 13 (e.g., paraffin oil), supplied from a single source, It is characteristic that the emerging droplets 15 are monodisperse (CVc2.5%), have volumes less than 10 nL, and do not coalescence with each other. The droplets are collected in a widening channel 16.

Example 3 (comparative example) In a non-preferred example depicted schematically in Fig. 3, a droplet 18 residing in a wide channel 1] is being split directly at the junction 21 without prior elongation of the entire droplet or a part thereof 19. In the junction, the droplet is being split by perpendicular streams of immiscible liquid 20 (e.g., paraffin oil), supplied from a single source. It is characteristic that the emerging droplets 22 are not monodisperse (CV>5%), have volumes less than 10 nL, and do not coalescence with each other. The droplets are collected in a widening channel 23.

Example 4

In a preferred example of embodiment of droplet splitting in a junction it is possible to obtain highly monodisperse droplets with volumes less than 10 nL. Fig. 4 shows a plot presenting the droplet size distribution as a function of junction geometry used. Preferably, the droplet is entirely (Fig. 1) or partly (Fig. 2) narrowed prior to splitting in the junction.

Narrowing the droplet results in droplet splitting conditions approaching those of splitting a stream of continuous liquid at flow-focusing junction. The narrowing minimises the flow of continuous phase in spaces between the droplet and the channel -in particular in corners of a channel with square cross section that are not occupied by the droplet. The most preferable is such embodiment of the system wherein the entire droplet is being elongated -it allows for obtaining a monodisperse population of droplets with coefficient of volume variation (CV) less than 2%. In a system where the entire droplet is elongated and maintains similar curvature irrespective of how the splitting is advanced, the Laplace pressure stays on a constant level which results in a stable generation of droplets of small volume. Equally preferable is the use of geometry with a short narrowing where only a fraction of a droplet is being elongated -CV for such geometry is less than 2.5%. Non-preferable is, however, to use a geometry without narrowing (Fig. 3) -the population of produced droplets is polydisperse and characterised by a CV of volume greater than 10%.

ExampleS

In a preferred example of embodiment of droplet splitting in a junction it is possible to obtain highly monodisperse droplets with volumes less than 10 nL. Fig. 5 shows a plot presenting the droplet size distribution as a function of surfactant concentration in the droplet phase. Unexpectedly, it turned out that preferable droplet size distributions are obtained for low surfactant concentrations, significantly below 1%, most preferably below 0.1%. High surfactant concentration results in unstable splitting of a droplet in the initial and final part thereof. On the other hand, surfactant concentration should be high (greater than 2%) to prevent coalescence of the emerging droplets -preferably it is obtained by increasing the surfactant concentration in the stream cutting the droplet.

Example 6

In a preferred example of embodiment of droplet splitting in a junction with long narrowing (Fig. 1B) it is possible to obtain highly monodisperse droplets with volumes less than 10 nL. Fig. 6 shows a plot presenting the average size of droplets produced by splitting as a function of flow rate of the cutting stream. The most preferable splitting takes place when the ratio between the flow rate of the cutting stream 6 and the flow rate of large drops 1 is from 1.5:1 to 3:1. In the disclosed example the flow rate of the droplet phase is 2ml/h. Using low flow rates of the cutting stream results in a low number of droplets in population and relatively large volume of emerging droplets. Using high flow rates of the cutting stream results in an excessive amount of the continuous phase and lower frequency of droplet generation.

Example 7

In a preferred example of embodiment it is possible to split droplets in a flow-focusing microfluidic junction, and grouping and separation of emerging droplets with a volume less than 10 nL in separate populations, so called,,droplet libraries". According to Fig. 7, a large drop 24 is split in a flow-focusing junction 25, and emerging droplets are subsequently transported to a tubing 26 with circular cross section. The system comprises a side channel 27 from which a droplet on demand 28 is produced, said droplet being composed of a third fluid, e.g., air, immiscible both with the continuous phase and the droplet phase, thus providing physical protection to the population of small droplets against mixing with each other. Automation of the production process of droplets separating the droplet libraries allows one to avoid mixing of elements belonging to adjacent libraries. In addition, the transverse channels in this junction are supplied with oil, whereas the flow of the oil is controlled by an external valve. This allows to switch on the oil flow while the droplet 24 is being split, and to switch it off when the droplet does not flow through the junction. It allows for avoiding an excess of the continuous phase in a droplet library, which is preferable in view of its further incubation, transport, or detection of results of a possible reaction. Automated generation of one library lasts for 5 seconds, after which next droplet 29, possibly of different composition, is being split.

ExampleS

In a preferred example of embodiment, according to Fig. 8, it is possible to incubate and transport droplet libraries 31 in a tubing 30 with circular cross section (alternatively in a channel with circular cross section). Preferably, the libraries 31 are separated by air bubbles 32, or droplets of another fluid, immiscible both with the continuous phase and the droplet phase.

Example 9

Fig. 9: a microfluidic cartridge 101 integrating a set of T junctions 102 that via ports 103 are supplied with continuous liquid, and a collection of fluids used to form droplets, delivered through ports 104, 105, 106, a junction 107 allowing for merging droplets produced in T-junctions, a mixing section 108, and a supply channel 110 to the second microfluidic junction 113, where the droplets are split into smaller ones, they fall into the inlet channel 111 that comprises an additional junction 115. The junction 115 allows for forming droplets (or bubbles) of additional fluid delivered through port 114, so as to allow for separating the families (libraries) of small droplets.

Example 10

Fig. 10: a supplying channel delivering droplets 209 to a microfluidic junction 204 is divided into sections 201, 202, 203, so that the ratio of width (and height) before the narrowing (e.g., 207) and behind the narrowing is not greater than 3. Preferably, the angle 206 formed by the wall (or floor/ceiling) of the channel in the narrowing with the channel axis is not greater than 300, preferably is equal to 45°, and is less than 600. Preferably, the last segment 203 of the supplying channel accommodates the entire droplet 209. Preferably, the last segment is wider than the microfluidic junction 204 at the most three times, and the narrowing 208 has walls forming with the section axis 203 an angle less than 60°. The junction 204 allows for breaking up the droplet 209 into a library of small droplets 210 entering the outlet channel 205.

In preferred embodiments of the invention indicated above a flow focusing junction was used as the microfluidic junction. Alternatively, other microfluidic junctions known in the state of the art can be used, as for instance a T-junction, or any other junction connecting flow of the droplet 209 and a continuous fluid immiscible therewith.

Claims

<claim-text>Claims 1. A method for splitting droplets (1) in a microfluidic junction (7) comprising the stage of a) supplying the droplet (1) to be split, suspended in a carrier liquid, to a microfluidic junction (7) through a microfluidic channel (2,4, 5) and the stage of b) breaking up the droplet (1) to be split in a microfluidic junction (7) into smaller droplets (8) using a stream of cutting fluid (6), immiscible with the liquid the droplet (1) to be split is made of, characterised in that the said stage a) of supplying the droplet (1) to be split, suspended in a carrier liquid, to the microfluidic junction (7) through a microfluidic channel (2, 4, 5) comprises transporting the droplet (1) to be split through a narrowed section (4, 5) of the microfluidic channel, whereas: i) the length of the narrowed section (4, 5) of the microfluidic channel (2,4,5) is at least equal to the longest dimension of the cross section of the microfluidic channel (2) before the narrowing, preferably at least equal to a hundredfold of that dimension, and most preferably at least equal a thousandfold of that dimension, and ii) the longest dimension of the cross section of the narrowed section of the microfluidic channel (4, 5) is from 1 to 5 times less than the longest dimension of the cross section of the microfluidic channel (2) before the narrowing, preferably from 2 to 3 times less than that dimension.</claim-text> <claim-text>2. Method according to claim 1, characterised in that Hi) the cross section area of the narrowed section of the microfluidic channel (4, 5) is from 1 to 25 times less than the cross section area of the microfluidic channel (2) before the narrowing, preferably from 4 to 10 times less than the cross section area of the microfluidic channel (2) before the narrowing.</claim-text> <claim-text>3. Method according to claim 1 or 2, characterised in that iv) the ratio in which the maximal dimension of the cross section of the microfluidic channel (2, 4, 5) changes as a result of narrowing thereof, to the length of the microfluidic channel on which the change takes place, is not less than 1.15, and not greater than 3.46, and preferably is equal to
2.</claim-text> <claim-text>4. Method according to claim 1, 2 or 3, characterised in that the said narrowed section of the microfluidic channel is composed of a series of narrower and narrower subsections, preferably from two to ten subsections, whereas each two consecutive subsections fulfil conditions i) and ii) as set forth in claim 1, and preferably also condition iii) as set forth in claim 2 and/or condition iv), as set forth in claim
3.</claim-text> <claim-text>5. Method according to any of the foregoing claims, characterised in that the said microfluidic junction (7) is a flow-focusing junction, T-junction or any other junction allowing for breaking up droplets into smaller ones.</claim-text> <claim-text>6. Method according to any of the foregoing claims, characterised in that the said microfluidic channels (2, 4, 5) are channels with rectangular, square or circular cross sections.</claim-text> <claim-text>7. Method according to any of the foregoing claims, characterised in that the said droplet (1) to be split has a volume from 100 nL to 100 p1.</claim-text> <claim-text>8. Method according to any of the foregoing claims, characterised in that the said droplet (1) to be split is being split into minimum 100, more preferably minimum 1000, yet more preferably minimum 10000, yet more preferably minimum 100000, and most preferably minimum 1000000 smaller droplets (8).</claim-text> <claim-text>9. Method according to any of the foregoing claims, characterised in that a sequence of droplets (1) to be split is formed, and the stages a) and b) are repeated for each droplet (1) in such a sequence.</claim-text> <claim-text>10. Method according to claim 9. characterised in that the collections of smaller droplets (8) resulting from splitting of each droplet (1) to be split in the said sequence are drained to the drain channel (26) located behind the microfluidic junction (7), and the collections of smaller droplets (8) are separated from each other by separating droplets (28) produced from a third fluid, immiscible both with the fluid used for producing smaller droplets and with the carrier liquid.</claim-text> <claim-text>11. Method according to claim 10, characterised in that the said third fluid is air, or silicone oil, fluorinated oil, hydrocarbon oil, or mineral oil.</claim-text> <claim-text>12. Method according to any of the foregoing claims, characterised in that the ratio of the flow rate of the cutting stream (6) and the flow rate of the droplets (1) to be split is from 1.5:1 to 3:1.</claim-text> <claim-text>13. Method according to any of the foregoing claims, characterised in that the droplets (1) to be split contain surfactant in an amount less than 1% by weight, preferably less than 0.1% by weight, and the cutting stream (6) contains surfactant in an amount over 2% by weight.</claim-text> <claim-text>14. A system for splitting droplets in a microfluidic junction comprising a microfluidic junction (7) and a microfluidic channel (2,4, 5) for supplying droplets (1) to be split to the microfluidic junction (7), characterised in that the said microfluidic channel (2, 4, 5) for supplying droplets (1) to be split to the microfluidic junction (7) comprises a narrowed section of the microfluidic channel (4, 5), whereas: i) the length of the narrowed section of the microfluidic channel (4, 5) is at least equal to the longest dimension of the cross section of the microfluidic channel (2) before the narrowing, preferably at least equal to a hundredfold of that dimension, and most preferably at least equal a thousandfold of that dimension, and H) the longest dimension of the cross section of the narrowed section of the microfluidic channel (4, 5) is from 1 to 5 times less than the longest dimension of the cross section of the microfluidic channel (2) before the narrowing, preferably from 2 to 3 times less than that dimension.</claim-text> <claim-text>15. System according to claim 14, characterised in that iii) the cross section area of the narrowed section of the niicrofluidic channel (4, 5) is from 1 to 25 times less than the cross section area of the microfluidic channel (2) before the narrowing, preferably from 4 to 10 times less than the cross section area of the microfluidic channel (2) before the narrowing.</claim-text> <claim-text>16. System according to claim 14 or 15, characterised in that iv) the ratio in which the maximal dimension of the cross section of the microfluidic channel (2, 4, 5) changes as a result of narrowing thereof, to the length of the microfluidic channel on which the change takes place, is not less than 1.15, and not greater than 3.46, and preferably is equal to 2.</claim-text> <claim-text>17. System according to claim 14, 15 or 16, characterised in that the said narrowed section of the microfluidic channel is composed of a series of narrower and narrower subsections, preferably from two to ten subsections, whereas each two consecutive subsections fulfil conditions i) and ii) as set forth in claim 14, and preferably also condition iii) as set forth in claim 15 and/or condition iv), as set forth in claim 16.</claim-text> <claim-text>18. System according to any of the foregoing claims from 14 to 17, characterised in that the said microfluidic junction (7) is a flow-focusing junction, 1-junction or any other junction allowing for breaking up droplets into smaller ones.</claim-text> <claim-text>19. System according to any of the foregoing claims from 14 to 18, characterised in that the said microfluidic channels (2,
4. 5) are channels with rectangular, square or circular cross sections.</claim-text> <claim-text>20. System according to any of the foregoing claims from 14 to 19, characterised in that additionally, behind the said microfluidic junction (7), the system has a drain channel (26) and a side channel (27), connecting to the said drain channel (26) in a T-junction, whereas the said side channel (27) is used to supply the third fluid to the said 1-junction.</claim-text>