The invention relates to a device and a method for high throughput, on demand generation of droplets. The invention finds application in microfluidics and in performing chemical reactions in microvolumes.

Suspensions of microscopic droplets resulting from dispersing one liquid phase in another liquid phase - called emulsions - apart from being common in natu re ( biologic a l su bsta nces) a re used a lso in a n u m ber of ind ustria l applications including manufacturing of paints, cosmetics and drugs. Most cosmetic creams and lotions are emulsions, and their properties are direct functions of the degree of dispersion, droplet stability, and size distribution. Emulsions are also often used in drug manufacturing because of better delivery and absorption of active ingredients.

Nowadays, emulsions are manufactured in industrial scale with the use of emulsifiers, i.e. surface active agents that facilitate generation and increase stability of droplets by reducing surface tension between selected fluids. The dispersing is usually accomplished by intensive and vigorous mixing of two liquid components. In the case of aerosols (suspensions of liquid droplets in gas, e.g., in air) use is made of breaking up a stream of liquid into droplets as a result of an instability caused by surface tension (Rayleigh-Plateau instability).

The method allows for a high-throughput generation of emulsions, but the dispersion of droplet size exceeds 10%. In recent years, new applications in chemical microanalysis, biotechnology and microencapsulation of pharmaceuticals have created high demand for capabilities to produce droplets with precisely defined sizes. A high degree of control over the droplet volume is offered by microfluidic devices producing droplets with geometries of typical sizes of a few to a few hu ndreds micrometers. Many solutions have been reported. Joscelyne and Tragardh (Journal of Membrane Science, 2000,

1 09: 1 07-1 1 7) proposed to pu mp droplet phase into the continuous phase through a membrane with holes of about 10 μιτι in size, and obtained a droplet volume distribution with standard deviation σ = 10%. Van Dijke et al. (Lab on a Chip, 2009, 9, 2824-2830) obtained droplets with a diameter of about 7.5 μιτι and σ = 10% using multiple narrow slits between channels containing immiscible fluids. Likewise, Kobayashi et al. (Industrial & Engineering Chemistry Research, 2005, 44, 5852-5850) obtained droplets with a diameter of about 30 μιτι and a standard deviation somewhat less than 10% using holes between chambers containing different fluids. Many applications, however, in particular those in chemical analysis and synthesis, require higher precision that at present can only be provided by microfluidic devices based on intersecting microchannels.

Droplet formulation in microchannels is a spontaneous process that takes place when flows of two immiscible fluids are crossed with each other. The droplet generation mechanism depends essentially on the geometry of the junction. The most often used solutions are flow-focusing junctions [P. Garstecki ef al. Physical Review Letters, 2005, 94, 104501 ], or T-junctions [P. Guillot, A. Colin, Physical Review E, 2005, 72, 066301]. In the flow-focusing junctions the droplet phase is supplied from one channel, whereas the continuous phase is supplied from two channels focused at the spot where the droplets are produced. In the T-junctions, the droplet phase is swept away by a perpendicular flow of the continuous phase. Spontaneous droplet generation in a single junction guarantees a high reproducibility of droplets, it has also, however, significant drawbacks. Regardless of the junction type, the droplets are produced only for appropriately small flows of the droplet phase, whereas laminar flow of both phases in a form of two parallel streams is observed for large flows. Therefore, the throughput of a microfluidic device is significantly limited. In addition, the droplet volume and the ratio of their volume to the volume of the continuous phase depend on volumetric flow rates of both phases and cannot be controlled independently of each other. The consequence of the above dependence is also that the droplet volume is susceptible to fluctuations in the supplying flows - a highly undesirable fact in industrial applications. An attempt to solve the low throughput problem may be to multiply the flow into many parallel channels supplied from a single source [T. Nisisako, T. Torii, Lab on a Chip, 2008, 8, 287-293]. The solution does not eliminate, however, the remaining limitations related to the spontaneous droplet generation, and simply increases them as branches may adversely affect the monodispersity of droplets as a result of uneven flow distribution in individual channels [V. Barbier ef a/., Physical Review E, 2000, 74, 04030ό; W. Li ef a/., Soft Matter 2008, 4, 258]. An additional hindrance are fluctuations of the system supply pressure that are difficult to eliminate and translate into fluctuations of the flow rate of each phase, and therefore of the sizes of droplets produced in the system. A recently proposed [P. Korczyk et a/., Lab on a Chip 2011, 11, 173-175] application of tubes with high hydraulic resistance (Fig. 1) in a form of thin (about 100 μιτΊ in diameter) and appropriately long (about 0.5 m) steel capillaries supplying the fluids to the system in a certain range significantly reduces the effect of the fluctuations mentioned above on the system operation, it involves, however, limitations such as that appropriately higher input pressures must be used. In the first place, however, it does not eliminate the problem of systematic pressure changes (mostly drops) resulting from imperfections of supplying devices that ultimately lead to systematic changes in the droplet size in time.

In recent years, a number of devices automating the droplet generation process have been reported. In these devices, the flow of the droplet phase is controlled with a valve, either a pneumatic valve [Y. Zheng et a/., Lab on a

Chip, 2009, 9, 409; K. Churski ef a/., Lab on a Chip, 2010, 10, 521], or a piezoelectric valve [A. Bransky et a/., Lab on a Chip, 2009, 9, 516], or by means of an active micro-reservoir [J. Xu, D. Attinger, Journal of Micromechanics and

Microengineering, 2008, 18, 005020]. In the system reported by Churski et al.

(Polish patent application No. P-390251, unpublished to date), a valve was used also to control the flow of the continuous phase. The solution allowed for a full dynamic control both over the volume of the droplets and their volume fraction. The method allowed to form any sequence of droplets with preset sizes in a fully reproducible way, without substantial limitations on the volume ratios or their order.

According to the invention, the device for high-throughput, on-demand generation of droplets is characterised in that it comprises n>2 microchannels originating at the first pressure buffer and n corresponding microchannels, originating at the second pressure buffer, whereas the said microchannels meet in pairs in n microfluidic junctions.

Preferably, the said first pressure buffer is connected to the first fluid source through the first distribution channel, the first port, and the first valve, and the said second pressure buffer is connected to the second fluid source through the second distribution channel, the second port, and the second valve.

In a preferred example of embodiment, the said first distribution channel between the said first port and the said first pressure buffer has minimum one branching, and preferably from 2 to 10 branchings.

In further preferred example of embodiment, the said second distribution channel between the said second port and the said second pressure buffer has minimum one branching, and preferably from 2 to 10 branchings.

Preferably, the said first valve is connected to the said first port through a tube with high hydraulic resistance, preferably a capillary, and the said second valve is connected to the said second port through a tube with high hydraulic resistance, preferably a capillary.

Preferably, the said microfluidic ju nctions ( 1 6) are T-junctions or flow- focusing junctions.

I n a pa rtic u la rly preferred exa m p le of e m bodim ent, the device according to the invention has a sensor, preferably a camera, for monitoring the size of the d rop let generated in at least one of the said microfluidic junctions, connected directly or indirectly to at least one of the said valves. In one of the preferred examples of embodiment, the device additionally comprises n>2 microchannels originating at the third pressure buffer, con necting in pairs to the said n microcha n nels, prefera bly so that the connection results in n serially connected pairs of microfluidic T-junctions.

The present invention comprises also a method for high-throughput, on- demand generation of droplets, according to which the first fluid is passed through n>2 microchannels originating at the first pressure buffer, and the second fluid is passed through n corresponding microchannels, originating at the second pressure buffer, whereas the said microchannels meet in pairs in n microfluidic junctions, a nd the flow of the first fluid is controlled by the first pressure buffer, a nd that of the second fluid is controlled by the second pressure buffer.

Preferably, the said first fluid, or the said second fluid is a fluid composed of two miscible or immiscible fluids.

Preferably, in the method according to the present invention droplets of two miscible fluids are generated sim u lta neously, a nd su bseq uently are merged, for instance using electric field, or with other methods of droplet coalescence known in the state of the art.

In a preferred example of embodiment, the said first pressure buffer is connected to the first fluid source through the first distribution channel, the first port, and the first valve, the said second pressure buffer is connected to the second fluid source through the second distribution channel, the second port, and the second valve, and the said control of the flow of the first fluid by the first pressure buffer and that of the second fluid by the second pressure buffer is accomplished by opening and closing the said first valve and the said second valve, respectively.

In a particularly preferred example of embodiment, behind at least one of the said microfluidic junctions a sensor is placed to monitor the size of droplets in that junction, preferably a camera, and the signal from the sensor is used to control operation of at least one of the said valves. Detailed description of the invention

Preferred examples of embodiments are now explained with reference to the accompanying figures, wherein:

Fig.1 shows a schematic diagram of connections of the elements that are external to the system, in particular the placement of reservoirs with fluids, valves and hydraulic tubes,

Fig.2 presents a schematic diagram illustrating the arrangement of the channels in the system together with the inlets and the outlet, and with labelled T-junctions,

Fig.3 presents a characteristic of the system operation with 16 parallel junctions without valves (a) and with valves (b),

Fig.4 shows the dependence of the droplet size on the flow rate of the droplet phase in a system with 16 parallel junctions with triggering of the droplet valve closing operation by the camera (,,·" symbols) and without triggering (,,o"parallel junctions), and

Fig.5 presents a schematic diagram of a single junction and a system of parallel connected junctions for a single T-junction (a) and a double T-junction (b), and in the case when two miscible droplet phases are used (c).

The essence of the present invention is the use of the solution proposed by Churski et al. (Polish patent application No. P-390251, unpublished to date) for supplying an appropriately designed system of parallel connected channels. Unexpectedly it turned out to be possible, in spite of the problems mentioned above. The use of valves in a single junction extends the range of permitted flow rates of both phases as well as the range of volumes and volume fractions of produced droplets, and makes the above independent of each other, and allows for generation of any sequence of droplets with different volumes (Fig. 3). Due to an appropriately designed system for simultaneous generation of droplets in many parallel junctions (Fig. 2), and in particular due to an appropriate geometry and distribution of channels, the above characteristics for a single junction remain preserved in the case of multiple junctions, while the throughput of the device is increased proportionally to the number of junctions. Novel is also the use of a real time control over the droplet volume that can be used both in single and in parallel system (Fig. 4). The solution consists in providing a feedback to the valve controlling the droplet phase by the sig na l from the ca m era or a nother detection device providing information about the droplet volume.

Example 1

The system described here (Fig. 1 ) is composed of external pressurised sources of fluids 1 , 2, valves 3, 4, tubes with high hydraulic resistance 5, 6 and the microfluidic system 7. External reservoirs with fluids 1 , 2 are directly connected to the computer controlled electromagnetic valves 3, 4. These valves are opened and closed according to a preset protocol. The outlet of each valve is connected to one of the two long tubes with high hydraulic resistance 5, 6, which supply fluids directly to the system. Each fluid is pumped into the system (Fig. 2) through round inlet holes 8, 9 to wide (0.8 mm) distribution channels 10, 1 1 forming a branched network. The purpose of these branches is to distribute evenly pressure. The outlets of the distribution channels are connected to elongated reservoirs 12, 13 that act as pressure buffers (one for each fluid) . The same number of long (about 2-3 cm) parallel microchannels 14, 15 (0.2 mm wide) come out from both reservoirs. So, each microchannel 15 containing the continuous phase has a corresponding microchannel 14 containing the droplet phase. The microchannels corresponding to each other meet at T-junctions 1 6 that are distant from the pressure buffers and have each a microchannel 1 7 draining fluids to the junction, where both phases flow jointly, with one of them in a form of droplets. Both phases leave the system through an atmospheric outlet 18. A simplified schematic diagrams of the system and of a single T-junction are shown in Fig. 5a.

Droplet generation in junctions 16 is controlled by operation of the valves 3, 4. The droplet phase is turned on for the time td while the continuous phase is turned off. Next, the droplet phase is turned off for the time t _c and the continuous phase is turned on, and the cycle is completed. With this method, for volumetric flows qd and q _c , respectively, we obtain a sequence of identical droplets, each with a volume of Vd = qdtd,o _P en, separated by fragments of continuous phase with a volume of V _c = qct _c ,o _P en. Thus, the length of droplets L and the intervals L _c between them in channels can be controlled by changing opening times of the valves (Fig.3b), td,o _P en and t _c ,o _P en (abbreviated as td and t _c ), as opposed to the situation without valves (Fig.3a), where the quantities, L and Lc, are both functions of volumetric flows, qd and q _c . The device allows also for designing a sequence of droplets with any volumes by appropriately selecting the opening times of the valves {(tdi,t _c i), (td2,t _C 2),...,(tdN,t _C N)}. The accuracy of reproduction of the above sequence of opening times in the form of a sequence of droplet volumes is determined separately for each droplet by the ratio At/td, wherein At is a delay related to a finite full opening or full closing time of the valve. In particular, it holds also td, t _c > 2At.

The droplet volume fraction can be calculated as cp= qdtd _rOP en /(qdtd,o _P en+ q _c tc,open), which means that, similarly as for the volume of the droplets, it can be controlled independently of the flows by adjusting the opening times of the valves. The upper limit for the volume fraction for a given droplet volume V is determined by the delay At and is cp _ma x(V) = V/(V+2q _c At).

The design of the system, and in particular the use of tubes with high hydraulic resistance 5, 6, branched distribution channels 10, 11, pressure buffers 12, 13, and long supplying microchannels 14, 15 to the junctions 16, aims at an increased hydrodynamic resistance of the system, and consequently at minimised relative fluctuations of the resistance due to the presence of droplets in the system and imperfections in fabrication of the microchannels 10, 11, 14, 15. In this way, reduced fluctuations of the resistance translate into reduced fluctuations of the droplet size. Increasing hydrodynamic resistances reduce the effect of fluctuations, but do not make the size of the droplets independent of the applied pressures. This goal can be reached only with a feedback, i.e., when the operation of the valves depends on the momentary size of the droplets. The result of such feedback, and in particular the constant volumes of the generated droplets, irrespective of the flow rate of the droplet phase is demonstrated in Fig. 4. In the presented example of embodiment, the feedback consists in triggering the droplet valve to close with a signal from the camera at the time moment, when a droplet in the selected channel reaches the desired length. The brightness threshold is set for a selected pixel in the camera, at a distance L from the junction, and when the threshold is exceeded it signals the emergence of the droplet-external phase interface. Then the valve with the droplet phase is closed, the continuous phase is turned on and a droplet of a length L is generated. Due to the fact that the same valve is used to operate all parallel channels, droplets of identical length are produced simultaneously in all channels. Therefore, the method of device operation implies that the droplet monodispersity in time in a given channel is determined by the accuracy of the feedback operation. Due to the size of the channels 14, 15, however, an important role here plays also the design of the device, and in particular the precision of fabrication and the length of microchannels 14, 15, as well as the geometry of the pressure buffers 12, 13 inside the system, and the length of external tubes with high hydraulic resistance 5, 6.

In the example of embodiment presented here, the microfluidic system is composed of three polycarbonate sheets. Appropriately wide distribution channels 10, 1 1 , 0.8 mm wide, and microchannels 14, 15, 0.2 mm wide, together with the T-junctions 1 6 are milled in the upper and in the lower sheets, 2 and 5 mm thick, respectively. The medium sheet separates the channels in both adjacent sheets, covering them at the same time, and has appropriately positioned through holes. The system is bonded in a hydraulic press under pressure 1 ΜΡα and a† temperature 130°C after prior modification of each plate in oxygen plasma.

The use of two valves 3, 4, one 4 controlling the continuous phase, and another one 3 - controlling the droplet phase, to operate many parallel connected junctions may be extended for the case of a greater number of valves, i.e., when one needs to introduce more than two phases into the system. Introducing an additional phase to the system opens the possibility of, e.g., scanning the conditions of chemical reactions (single junction adapted for this purpose has been reported by K. Churski et al. in the Polish patent application No. P-390251, unpublished to date), or producing dual emulsions (an example of a passive system with single junction adapted for that purpose has been reported by N. Panacci et al., Physical Review Letters, 2008, 101, 104502).

Example 2

Below we describe an integrated system of multiple dual junctions (Fig. 5b) that could be used for production of dual emulsions with a throughput increased by many times. An additional, third phase is supplied to the system and distributed inside the system analogously as the two remaining phases. It is noteworthy, however, that addition of every next phase is related to an increase by two of the number of sheets the system is composed of, as it is necessary to avoid channel crossing. Depending on the intended use of the system, it is possible to use junctions of various types. An interesting example is a combination of two T-junctions that can be used to generate multiple droplets (in the case immiscible droplet phases are used). Such a system in an example of embodiment is illustrated in Fig.5b. Droplets of, for instance, phase C in phase B (junction TCB), i.e., C/B emulsion, are produced in the first T-junction, whereas in the second junction (TBA) droplets of phase B in phase A are generated, which corresponds to a dual C/B/A emulsion. Example 3

Below we describe an integrated system of multiple dual junctions (Fig. 5b) that could be used for production of mixtures, also with a throughput increased by many times. Here, the use is made of a dual T-junction of different type than the one used for generation of dual emulsions. Miscible droplet phases B and C are independently supplied to the continuous phase A using two junctions TCA and TBA, giving rise to a pair of droplets C/A and B/A. Such a pair of droplets can subsequently coalescence, for instance as a result of an applied electric field, generated by electrodes placed in the vicinity of the junction. A corresponding system in an example of embodiment is illustrated in Fig.5c. By changing the volumes of droplets B and C one gains control over the reagent ratios in a merged droplet B+C. The mixing efficiency of the components inside the merged droplet is assured by curved inlet channels. The movement of a droplet inside curved channels leads to a displacement of the swirls inside the droplet thus allowing for mixing [H. Song, J. D. Tice, R. F. Ismagilov, Angew. Chem. Int. Ed.2003, 42, 768].