Microfluidic device for the production of emulsion microdroplets and method to produce the emulsion microdroplets

Abstract

Microfluidic device (10) for the production of emulsion microdroplets, comprising a first microfluidic channel (20), a second microfluidic channel (30) being fluidly connected at a first junction (40) to the first microfluidic channel (20), a main channel (50) extending from the first junction (40) to a main channel end (54), the first microfluidic channel (20) being designed to carry a first fluid (60) and the second microfluidic channel (30) being designed to carry a second fluid (70), the second fluid being immiscible with the first fluid to develop a flow with a flow profile such that the first fluid is surrounded by the second fluid in the main channel, and further comprising means to control the flow velocity of the first fluid and the second fluid to allow the generation of emulsion microdroplets (92) of the first fluid in the main channel, wherein a cooling means (100) designed to cool at least a portion of the main channel (50) to form a cooling area through which, in use, emulsion microdroplets (92) are transported for cooling the emulsion microdroplets, characterized in that the main channel (50) is fluidly connected downstream of the cooling area to a third microfluidic channel (120) at a main junction (122), wherein at least a portion of the main channel end (54) protrudes in the third microfluidic channel and forms a passage (124) between the main channel end (54) and the wall of the third microfluidic channel facing the main channel end, the width (154) of the passage being measured radially to the third microfluidic channel as the distance between a foremost end (150) of the main channel end (54) and the wall of the third microfluidic channel (120) facing the foremost end, the third microfluidic channel being designed to carry a further fluid (126), the second fluid (70) being immiscible with the further fluid (126) to develop a flow with a flow profile such that the further fluid is surrounded by the second fluid in the third microfluidic channel downstream of the main junction (122), and further comprising means to control the flow velocity of the further fluid to form double-emulsion microdroplets downstream of the main junction (122), the double-emulsion microdroplets having a core formed by an emulsion microdroplet of the first fluid surrounded by the further fluid.

Description

[0001] Microfluidic device for the production of emulsion microdroplets and method to produce the emulsion microdroplets .

[0002] The present invention generally relates to microfluidic devices and methods of producing microdroplets , including single other multiple emuls ion microdroplets .

[0003] In the case of double emulsion microdroplets , each microdroplet contains one smaller drop that is composed of a fluid di f ferent to the fluid forming the rest of the microdroplet . These coreshell structured fluids can be used, for instance , as templates to produce capsules . The outer drop contains the material that forms the capsule shell , whereas the inner drop constitutes the capsule core . These capsules can be used as vehicles for delivery of active ingredients in many fields , such as food, pharmaceuticals , or cosmetics .

[0004] More speci fically in the field of pharmaceuticals , classical drug delivery in the human body relies on systemically administering the therapeutic agent into the bloodstream to be distributed in the entire body and thus reach the target site . While this is an ef fective strategy, most therapeutic agents do not reach the target site but generate of f-target ef fects . Medical profes sionals must make a tradeof f : a higher dose can increase the therapeutic ef fect but harm the patient . Conversely, a lower amount reduces side ef fects but may also be less ef fective . Targeting the release to a speci fic location enables a much higher local concentration while eliminating most side ef fects . In further application, the system can allow for a more ef ficient delivery of complex therapeutics like cells and gene therapies that could otherwise be disrupted by the immune system .

[0005] However, success ful application of these capsules may require good control over their permeability and mechanical stability, parameters that can be tuned with the composition and thickness of the capsule shell . This may involve control over the dimensions and composition of the double emulsions . This control is often di f ficult to achieve i f double emulsions are produced by mechanical stirring or membrane emulsi fication, since these conventional approaches typically yield double emulsion drops of di f ferent si zes that often contain multiple inner microdroplets .

[0006] WO2015 / 160919 discloses microfluidic systems and methods of producing microdroplets . In one embodiment , a device includes a first fluid flowing through a first channel towards a first j unction . At the first j unction, the first channel is intersected by a pair of second channels through which a second fluid can flow, the second fluid being immiscible with the first fluid . Thus , a flow profile of the first fluid substantially surrounded by the second fluid may develop in a further channel extending from the first j unction . The further channel may have portions having di f ferent cross-sectional areas . The first and second fluids flowing through the further channel enter a second j unction formed at the intersection of the further channel and of a pair of third channels in which a third fluid flows , the third fluid being at least immiscible with the second fluid . The introduction of the third fluid into the second j unction can cause the second fluid and the first fluid to form individual discrete microdroplets within the third fluid . The microdroplets can be double emulsion microdroplets comprising a microdroplet of a first fluid surrounded by a microdroplet of the second fluid, which is in turn surrounded by the third fluid . By controlling the volumes and / or flow rates of the various fluids , the volumes and / or thickness of the components of the microdroplets may be controlled . However, according to this method, two successive layers of the microdroplets must be made starting from fluids that are not miscible with each other to allow the formation of a flow profile leading to the generation of microdroplets . For example , the formation of a microdroplet having two successive layers of aqueous substances , e . g . two layers of gelatine having di f ferent composition, would not be possible .

[0007] US2012211084A1 relates to multiple emulsions formed by urging a fluid into a channel , e . g . , by causing the fluid to enter the channel as a j et . Side channels can be used to encapsulate the fluid with a surrounding fluid . Multiple droplet may be formed from the collinear flow of fluids at or near a single location within the fluidic channel . In figure 1A, a device is disclosed comprising a main channel , which can be a microfluidic channel . Intersecting the main channel are a plurality of side channels , speci fically a first set of channels that intersects the main channel to define a first intersection, and a second set of channels that intersects the main channel to define a second intersection . An inner fluid may be delivered to the device through the main channel , while an outer fluid can be delivered through the first set of channels , meeting the main channel at the first intersection . At the second intersection, an outer fluid may be delivered via the second set of channels . The carrying fluid may surround the inner and outer fluids , in some cases causing the inner and outer fluids to form multiple emulsion droplets where the outer fluid surrounds the inner fluid . W02009048532A2 relates to emulsions and, in particular, to systems and methods for forming multiple emulsions and emulsions produced therefrom . In an embodiment , a microfluidic device includes a T- j unction . A multiple emulsion can be formed by directing a fluidic droplet within a first channel , containing an inner fluid, at a non-linear intersection of the first and a second channel . The second channel may contain a second fluid that is substantially immiscible with the fluidic droplet . Upon entering the second channel , the fluidic droplet may be encapsulated by the second fluid, thereby forming a multiple emulsion . In an example shown in Fig . 1C, a gas is contained within a water droplet , which in turn is directed into an oil phase .

[0008] Consequently, there is still a need to develop further devices and methods for producing microdroplets , including single and multiple emulsions microdroplets , in particular that allow the production of microdroplets made of successive layers formed by substances that are miscible .

[0009] The obj ect of the present invention is to provide a microfluidic device and a method for the production of emulsion microdroplets that allow the production of microdroplets made of successive layers . In particular, the succes sive layers can be made of substances that are miscible without requiring an intermediary separation layer between the miscible substances during the production of the microdroplets and in the microdroplets . In addition, the device and the method allow the production of microdroplets which layers have di f ferent composition and contain agents like therapeutic agents , contrast agents or the like . The problem is solved by a microfluidic device for the production o f emulsion microdroplets according to claim 1 and a method for the production of emulsion microdroplets according to claim 6 . Preferred embodiments are disclosed in the dependent claims .

[0010] In a first aspect , the present invention is directed to a microfluidic device for the production of emulsion microdroplets . The microfluidic device comprises a first microfluidic channel , a second microfluidic channel fluidly connected at a first j unction to the first microfluidic channel and a main channel extending from the first j unction to a main channel end . At the first j unction, the first and second microfluidic channels can be fluidly connected for example in the form of an intersection or a common opening or other means .

[0011] The first microfluidic channel is designed to carry a first fluid and the second microfluidic channel is designed to carry a second fluid, the second fluid being immiscible with the first fluid to develop a flow with a flow profile such that the first fluid is surrounded by the second fluid in the main channel . Thus , little or no mixing of the first and second fluid may occur at the first j unction .

[0012] In a preferred embodiment , the inner surface of the main channel may be hydrophobic, either as a result of a surface treatment or a choice of material for the main channel having properties . This facilitate having the second fluid closer to walls of the main channel and the first fluid farther away from the walls and towards the center of the main channel , for example i f the first fluid is rather hydrophilic and the second fluid is relatively hydrophobic . An opposite configuration is also possible in other embodiments . In a preferred embodiment , the microchannels have a constant cross-section over their respective length to allow a simple construction with standard parts . However, it is also possible to have portions having di f ferent cross-sectional areas , for example a change in cross-sectional area in the main channel may be used to cause the first and the second microfluid to flow faster through the main channel to influence the formation and the si ze of emulsion microdroplets .

[0013] In a preferred embodiment , the cross-sectional area of the main channel measured at the first j unction corresponds to , preferably is larger than the sum of the cross-sectional area of the first and second microfluidic channel to limit backflow in the first and second microfluidic channel .

[0014] In a preferred embodiment , the first and second microfluidic channel may be fluidly connected, forming an intersection of the two channels at the first j unction at a j unction angle of 90 ° . Intersection at a j unction angle di f ferent to 90 ° may also be possible .

[0015] The microfluidic device further comprises means to control the flow velocity of the first fluid and the second fluid to allow the generation of emulsion microdroplets of the first fluid in the main channel . By controlling the flow velocities , the volume of the emulsion microdroplets can be controlled .

[0016] According to the invention, the microfluidic device comprises a cooling means designed to cool at least a portion of the main channel to form a cooling area through which, in use , emulsion microdroplets are transported for cooling the emulsion microdroplets . The cooling means are designed to achieve a cooling temperature at which a hardening of the emulsion microdroplets of the first fluid takes place . In this state , the emulsion microdroplets can be collected, i f necessary stored, and further processed .

[0017] In the present disclosure , an emulsion microdroplet is a microdroplet of a fluid dispersed in another fluid, a so called carrying fluid . The emulsion microdroplet can be formed as a shell that contains one inner microdroplet of the same fluid, said same fluid carrying for example particles or agents that are not present in the shell fluid, or another fluid . The shell can contain more than one inner microdroplets each of a di f ferent fluid or of the same fluid, wherein for example an outermost inner microdroplet surrounds a further inner microdroplet . The carrying fluid is typically substantially immiscible with the microdroplet of fluid . In addition, the fluid within the inner microdroplets can be miscible or substantially immiscible with the fluid of shell . For instance , the innermost fluid may be an aqueous fluid and the outer fluid forming the shell may be a lipophilic phase that is substantially immiscible with the aqueous fluid . In another instance , the innermost fluid may be an aqueous fluid and the outer fluid forming the shell may be also an aqueous phase that is miscible with the aqueous fluid . Furthermore , these principles may be extended to higher-order multiple emulsions microdroplets .

[0018] As used herein, two fluids are immiscible , or not miscible , with each other when one is not soluble in the other to a level of at least 10% by weight at the temperature and under the conditions at which the emulsion is produced . For instance , two fluids may be selected to be immiscible within the time frame of the formation of the fluidic microdroplets . In some embodiments , two fluids ( e . g . , the carrying fluid and the inner microdroplet fluid of a multiple emulsion) are compatible , or miscible , while the outer microdroplet fluid is incompatible or immiscible with one or both of the carrying and inner microdroplet fluids . In other embodiments , however, all three fluids may be mutually immiscible , and in certain cases , all of the fluids do not all necessarily have to be water soluble .

[0019] As used herein, the term " fluid" generally refers to a substance that tends to flow and to conform to the outline of its container, i . e . , a liquid, a viscoelastic fluid, etc . The term fluid does not refer to a substance in the form of a gas . Typically, fluids are materials that are unable to withstand a static shear stress , and when a shear stress is applied, the fluid experiences a continuing and permanent distortion . The fluid may have any suitable viscosity that permits flow .

[0020] In this regard, it is noted the flow velocity of a fluid and the volume flow of this fluid are physically linked . This is also the case for the pressure di f ference under which the fluid flows . Therefore , the reference to a control of the flow velocity can be considered as equivalent to the control of the volume flow or a pressure di f ference in the present context , wherein the technical means to measure and control the parameters may be di f ferent . Some microfluidic pumps can regulate the volume flow, others regulate the velocity, and some a pressure di f ference . In any case , all these parameters are intrinsically connected . For example , volume flow or flow velocity are the same with a factor of cross-sectional area of the channel .

[0021] Hardening of the emulsion microdroplets refers to an increase of the resistance of the microdroplets to deformation . Hardening of the emulsion microdroplets by cooling can be the result of one or more physical processes that take place simultaneously or sequentially depending on the nature of the first microfluid and / or the second microfluid. Most of the methods using UV curing rely on acrylates that are not biocompatible. Further, in embodiments in which a large quantity of magnetic nanoparticles is present, it is difficult to cure the UV-curable polymer, as the light is absorbed by the nanoparticles. Thus, thermal curing is a simple and nontoxic way of achieving hardening of the emulsion microdroplets.

[0022] In an embodiment, the first microfluid can be a sol-gel, i.e. a material that can be in a sol or a gel state, and typically includes polymers. The gel state typically contains a polymeric network containing a liquid phase, and can be produced from the sol state by removing solvent from the sol, e.g., via drying or heating techniques. In some cases, the sol may be pretreated before being used, for instance, by causing some polymerization to occur within the sol. By cooling emulsion microdroplets of the first microfluid a sol-gel transition can be initiated such that it becomes harder. More generally, cooling can initiate a physical or a chemical crosslinking that results in emulsion microdroplets becoming harder. It is also conceivable that hardening is the result of change of viscosity .

[0023] "Microfluidic, " as used herein, refers to a device, apparatus, or system including at least one fluid channel having a cross- sectional dimension of less than about 1 millimeter (mm) , thereby providing microdroplets having comparable average diameters. One or more channels of the system may be a capillary tube. In some cases, multiple channels are provided.

[0024] In the present case, the device, apparatus, or system can also have a cross-sectional dimension in the millimeter range, a field to which it is also referred to as small scale fluidics . Depending on the si ze of the emulsion droplets required, cross- sectional dimens ion up to 3 mm are possible , this value being not limiting . For the sake of simplicity, the term "microfluidic" will be consistently used to cover an embodiment in which the relevant cross-section is in the millimeter range .

[0025] The same applies to the term microdroplet to refer to droplet having a diameter of less than about 1 millimeter (mm) or more than 1 millimeter as it is the case for droplets produced by the device , apparatus , or system having a cross-sectional dimension in the millimeter range and to which it is also referred by the term "microfluidic" by extension, as explained above .

[0026] One or more of the channels may, in cross-section, have a height that is substantially the same as a width at the same point . In cross-section, the channels may be circular, rectangular, or substantially non-rectangular , such as circular or elliptical .

[0027] Further according to the invention, the main channel is fluidly connected downstream of the cooling area to a third microfluidic channel at a main j unction . At least a portion of the main channel end protrudes in the third microfluidic channel and forms a passage between the main channel end and the wall of the third microfluidic channel facing the main channel end . As a result , the passage has a reduced crosssection compared to the cross-section of the third microfluidic channel . The width of the passage is measured radially to the third microf luidic channel as the distance between a foremost end of the main channel end and the wal l of the third microfluidic channel facing the foremost end . The third microfluidic channel is designed to carry a further fluid, the second fluid being immiscible with the further fluid to develop a flow with a flow profile such that the further fluid is surrounded by the second fluid in the third microfluidic channel downstream of the main j unction . In addition, the microfluidic device comprises means to control the flow velocity of the further fluid to form double-emulsion microdroplets downstream of the main j unction . The doubleemulsion microdroplets have a core formed by an emulsion microdroplet of the first fluid surrounded by the further fluid .

[0028] The further fluid can also be substantially surrounded by the second fluid in the third microfluidic channel in the trans fer zone , for example in case turbulences are present . This arrangement allows a trans fer of the emulsion microdroplets into the further fluid . It also allows the development of a flow in the third microfluidic channel downstream of the main j unction with a flow profile such that the further fluid containing the emulsion microdroplets is surrounded by the second fluid . By controlling the flow velocity of the further fluid, it is possible to generate downstream of the main j unction double-emulsion microdroplets having a core formed by an emulsion microdroplet of the first fluid surrounded by the further fluid in the form of a shell made of the further fluid . As result the shell forms a coating made of the further fluid directly applied onto the emulsion microdroplet , i . e . the double-emulsion microdroplet is free from a layer separating the first fluid and the further fluid . In the present context , " free" is to be understood such that residues of the second fluid could be captured due to turbulences during the encapsulation o f the first fluid by the further fluid and might be present locally without however forming a continuous separation layer . Therefore , it is " free" within the limits of the physical process that is happening . More speci fically the hardened emulsion microdroplets allow processing in such a manner that they do not mix with the further fluid before forming double-emulsion microdroplets . In the same manner, multiple emulsions microdroplets , also referred to as multiemulsion microdroplets , can be formed by repeating the same procedure .

[0029] The trans fer of the emulsion microdroplets into the further fluid and the following formation of the shell around the emulsion microdroplets is achieved in a simple manner after the core made of the first fluid has been hardened previously in the cooling area . This allows a mechanical shi fting of the emulsion microdroplet in the further fluid . Here , constraint in the third microfluidic channel is used to mechanically push the emulsion microdroplet from the second fluid into the further fluid . Further, the means to control the flow velocity of the further fluid allows to control the volume of the double-emulsion microdroplets and in particular the thickness of the shell .

[0030] In a preferred embodiment , the third microfluidic channel and the main channel may intersect , i . e . are fluidly connected, at the main j unction at an intersection angle of 90 ° for a simple arrangement . However, intersection at a non-right angle may also be possible . In particular, the intersection angle might be smaller than 90 ° , for example between 60 ° and 90 ° , preferably between 75 ° to 85 ° , to reduce turbulences at the intersection, thereby improving the trans fer of the emulsion microdroplets in the further fluid . In a preferred embodiment , the main channel end extends in a transversal plane at an angle with respect to a longitudinal axis of the main channel , thereby forming, as seen in a longitudinal cross section of the main channel , a foremost end of the main channel and an of fset end of the main channel arranged upstream of the foremost end . At least a portion of the main channel end protrudes in the third microfluidic channel to form the passage between the foremost end and the wall of the third microfluidic channel facing the foremost end . The width of the passage is measured radially to the third microfluidic channel as the distance between the foremost end and the wall of the third microfluidic channel facing the foremost end . Further, the transversal plane is oriented such that its normal facing away from the main channel has a component that is oppositely directed to a direction of flow of the further fluid in use .

[0031] In other words , the main channel end is formed as a single bevel corresponding to the transversal plan . For example , in the case of cylindrical main channel , the main end channel extends in the transversal plane in the form of an ellipse . Such a transversal plan can advantageously be obtained by cutting the main channel at an angle with respect to the longitudinal axis of the main channel .

[0032] In a preferred embodiment , the angle with respect to the longitudinal axis of the main channel is between 20 ° and 60 ° , preferably between 30 ° and 50 ° . The si ze of the angle is directly connected to the si ze of an opening surface at the end of the main channel . The opening surface forms a surface that intersects at least partially the further fluid flow, thereby forming an ef ficient trans fer zone in which or in the vicinity of which the further fluid flow is constricted in such a manner that the trans fer of emulsion microdroplets into the further fluid takes place . The angle can be chosen depending on the si ze of the trans fer zone necessary to optimi ze the trans fer . For the simplicity of the arrangement and flow calculation, an angle of 45 ° is most preferred .

[0033] Preferably, the longitudinal axis of the main channel intersects the longitudinal axis of the third microfluidic channel to provide for a trans fer of the emulsion microdroplets in a centered manner with respect to the direction of flow of the further fluid in the third microfluidic channel . The symmetry of the arrangement allows an improved centering of the emulsion microdroplet in the further fluid, which in turns can have an influence on the final centering of the core within the double emulsion microdroplet .

[0034] In use , the flow of the further fluid is reduced, as seen in cross-section, to the passage left by the main channel intersecting the third microfluidic channel . The shape of the passage depends on the cross-section of the main channel and of the third microfluidic channel at the intersection .

[0035] In a preferred embodiment , the first , second and third microfluidic channel as well as the main channel have a circular cross-section to take advantage of the symmetry for the formation of co- flow . However, elliptical cross-section can also be used . In other cases , a square cross-section may be used .

[0036] In an embodiment in which the main channel has a rectangular cross section and the third microfluidic channel has a circular cross-section in the form of a disk, wherein the foremost end is a side of the rectangular cross section that corresponds to a chord of the disk, i . e . the endpoints of the side correspond to the endpoints of a chord of the disk, the passage has the form of the disk with a flat side . In a similar embodiment in which the main channel is introduced further in the third microfluidic channel , i . e . the foremost end extends farther in the third microfluidic channel , the passage has the form of the disk in which a rectangular recess is present .

[0037] A characteristic of the passage that is relevant for the trans fer of emulsion microdroplets into the further fluid is the distance between the foremost end and the wall of the third microfluidic channel facing the foremost end . The width of the passage is measured radially to the third microfluidic channel , i . e . in a direction perpendicular to the flow direction of the further fluid at the intersection . In case the distance between the foremost end and the wall of the third microfluidic channel facing the foremost end can be measured at di f ferent points of the foremost end, the width of the passage is defined as the maximum width measured . The width of the passage can play a role in dimensioning the si ze of the double-emulsion microdroplets . In one embodiment , for example , the passage can be dimensioned such that the emul sion microdroplets are squeezed while they are trans ferred into the further fluid and pass through the passage , thereby leaving less space for the passage of further fluid that is available to form the shell . The result can be a thinner shell .

[0038] The normal to the transversal plane can be defined as a line extending perpendicularly to the transversal plane . Such a line can extend in two directions with respect to the transversal plane . Presently, the normal of interest is facing away from the main channel , i . e . the normal is facing towards the outside of the main channel . To ensure that the further fluid meets the second fluid transporting the emulsion microdroplets in the trans fer zone , it is necessary that the further fluid meets at least a portion of the opening surface . This condition is met when a component of the normal facing away from the main channel is oppositely directed to the direction of flow of the further fluid, when the microfluidic device is in use .

[0039] In a preferred embodiment , the width of the passage is larger, preferably 25% , more preferably 10% larger than the average diameter of the emulsion microdroplets . The larger passage avoids squeezing the emulsion microdroplets and forms a space for the passage of further fluid to allow the formation of a thickener shell around the core . A width larger by 10% has shown experimentally stable shell thickness . This arrangement is preferred for single emulsions . Without being bound by the theory, it is believed that this value allows a reduction of turbulences at the intersection so that a more stable flow of further fluid and consequently of shell thickness is possible .

[0040] In a preferred embodiment , the width of the passage is smaller, preferably 30% smaller, more preferably 20% smaller than the average diameter of the microdroplets . In contrary to the previous embodiment , the smaller passage results in squeezing the emulsion microdroplets and constricting the flow of further fluid . Less space is available for the passage of further fluid that is available to form the shell so that the cross-section of the further fluid flow in which the emulsion microdroplets are captured is smaller . It follows that a thinner shell can be obtained . This arrangement is preferred for multiple emulsions microdroplets .

[0041] In a preferred embodiment , the main j unction is arranged in an interface device having a further fluid inlet designed to receive a portion of the third microfluidic channel extending upstream of the main j unction, and an outlet designed to receive a portion of the third microfluidic channel extending downstream of the main j unction . The outlet is connected to the further fluid inlet by an interface channel . The interface device has further a receiving channel fluidly connected to the interface channel and designed to receive the main channel end . The interface device has the advantage that the geometry of the main j unction, i . e . the relative position of the main channel end and of the third channel , can be set and formed in a fixed manner . As a result , a fine tuning of the arrangement of the main channel end and of the third channel is not necessary each time a production of double-emulsion microdroplets is started . The main channel can be inserted in a fluid-tight manner into the receiving channel acting as a guide and the upstream portion of the third microfluidic channel supplying the further fluid can be simply inserted in a fluid-tight manner into the further fluid inlet . Further, the downstream portion of the third microfluidic channel can be inserted in a fluid-tight manner into the outlet to allow a flow downstream of the j unction, wherein the upstream portion and the downstream portion of the third microfluidic channel are connected to each other by the intermediate portion of the third microf luidic channel present in the interface device to form altogether the third microfluidic channel . Further, the provision of a fluid-tight connection at the j unction can be di f ficult because of its geometry . Advantageously, the interface device in which the j unction can be embedded ensures the fluid-tight connection of the corresponding channels at the j unction, whereas the connections in a fluid-tight manner at the outlet and inlets of the interface device are less challenging . In another preferred embodiment , the main channel end is formed as a fixed part at the end of the receiving channel facing the intermediate portion, in which case the form of the passage at the j unction is also set in a fixed manner . In this embodiment , an upstream portion of the main channel can be simply inserted into the receiving channel to provide for a fluid connection with the main channel end . The main channel end can be formed and / or positioned relatively to the third microfluidic channel according to any of the main channel end embodiments described in which the interface device is not provided for .

[0042] In a preferred embodiment , the interface channel widens before the outlet of the interface device to improve the formation of the outer microdroplets .

[0043] In a preferred embodiment , the interface device can be molded or produced by additive manufacturing as a solid block in which the j unction and the corresponding channels are embedded . Typically, polydimethylsiloxane ( PDMS ) can be used among other materials that are commonly used in the field of microfluidics . Other production process can be used like patterning a material blank by one of many forms of lithography to design the channels and etching the corresponding patterns in a subsequent step to form the channels , using processes like those used in microsystems (MEMS ) production .

[0044] Preferably, the microfluidic device comprises means to mechanically adj ust the width of the passage , i . e . the depth of insertion of the main channel end, in the third microfluidic device in the longitudinal direction to adj ust the width of the passage . Such means can be for example a linear guide or a carriage to which the main channel is attached . In a second aspect of the invention, a method to produce emulsion microdroplets is disclosed, comprising the steps of a ) providing a microfluidic device according to any one of the embodiments disclosed in the first aspect of the invention; b ) controlling the flow velocity of the first f luid and of the second fluid to generate emulsion microdroplets of the first fluid in the main channel , the second fluid being immiscible with the first fluid, and c ) cooling at least a portion of the main channel to harden the emulsion microdroplets .

[0045] Advantageously, the method allows the production of hardened emulsion microdroplets made of the first fluid . In this state , the emulsion microdroplets can be collected, i f necessary stored, and further processed .

[0046] The relative flow velocities of the first fluid and second fluid are controlled such that microdroplets of the first fluid can be generated in the main channel .

[0047] Di f ferent methods are possible to generate microdroplets . Coflow is a droplet formation method in which the first fluid is guided through a channel , here the first microfluidic channel , that is enclosed inside a channel in which a second fluid is guided, here the main channel by way of the second microfluidic channel . In the main channel , the first fluid is stretched until it breaks from shear forces and forms microdroplets either by dripping or j etting .

[0048] Co- flow can be implemented with a dripping regime when the flow velocities of both the first and second fluid are low, wherein microdroplets of the first fluid are produced due to surface tension and set apart from the first fluid under the drag force of the second fluid . Microdroplets of the first fluid can also be generated when co- flow is implemented with a j etting regime in which the first fluid flows faster than the second fluid, causing a j et of the first fluid to expand and break up followed by the formation of spherical microdroplets of the first fluid .

[0049] The co- flow in the microfluidic device has the advantage that the first fluid is wrapped by the second fluid, thus the microdroplets are formed in a three-dimensional environment that improves the regularity of their shape , and in which the surface wettability of the channels , in particular the main channel has less influence on the formation of the emulsion microdroplets .

[0050] However, other methods of microdroplet generation could also be used, such as T-Junction or flow- focusing to which it is also sometimes referred as a co- flow in the literature .

[0051] The microfluidic device provided is further such that the main channel is fluidly connected downstream of the cooling area to a third microfluidic channel at a main j unction . At least a portion of the main channel end protrudes in the third microfluidic channel and forms a passage between the main channel end and the wall of the third microfluidic channel facing the main channel end . As a result , the passage has a reduced cross-section compared to the cross-section of the third microfluidic channel . The width of the passage is measured radially to the third microfluidic channel as the distance between a foremost end of the main channel end and the wall of the third microfluidic channel facing the foremost end . The third microfluidic channel is designed to carry a further fluid to form double-emulsion microdroplets downstream of the main j unction . The double-emulsion microdroplets have a core formed by an emulsion microdroplet of the first fluid surrounded by the further fluid . In addition, the microfluidic device further comprises means to control the flow velocity of the further fluid .

[0052] Further, the method compri ses the further steps of d) controlling the flow velocity of the further fluid, the second fluid being immiscible with the further fluid, to trans fer the emulsion microdroplets from the second fluid into the further fluid and develop a flow in the third microfluidic channel downstream of the passage with a flow profile such that the further fluid containing the emulsion microdroplets is surrounded by the second fluid to allow the generation of double-emulsion microdroplets downstream of the passage , the double emulsion microdroplets having a core formed by an emulsion microdroplet of the first fluid surrounded by the further fluid .

[0053] This step allows a trans fer of the emulsion microdroplets into the further fluid and the development of a flow in the third microfluidic channel downstream of the intersection . The flow profile is such that the further fluid containing the emulsion microdroplets is surrounded by the second fluid . By controlling the flow velocity of the further fluid, it is possible to generate downstream of the intersection double-emulsion microdroplets having a core formed by an emulsion microdroplet of the first fluid surrounded by the further fluid in the form of a shell made of the further fluid . As result the shell forms a coating made of the further fluid directly applied onto the emulsion microdroplet , i . e . the double-emulsion microdroplet can be free from a layer separating the first fluid and the further fluid . More speci fically the hardened emulsion microdroplets allow processing in such a manner that they do not mix with the further fluid before forming double-emulsion microdroplets .

[0054] The trans fer of the emulsion microdroplets into the further fluid and the following formation of the shell around the emulsion microdroplets is achieved in a simple manner after the core made of the first fluid has been hardened previously in the cooling area . This allows a mechanical shi fting of the emulsion microdroplet in the further fluid . Here , constraint in the third microfluidic channel is used to mechanically push the emulsion microdroplet from the second fluid into the further fluid . Further, the means to control the flow velocity of the further fluid allows to control the volume of the double-emulsion microdroplets and in particular the thickness of the shell .

[0055] In a further preferred embodiment , the main channel end extends in a transversal plane at an angle with respect to a longitudinal axis of the main channel , and thereby forms , as seen in a longitudinal cross section of the main channel , the foremost end of the main channel and an of fset end of the main channel arranged upstream of the foremost end . At least a portion of the main channel end protrudes in the third microfluidic channel to form the passage between the foremost end and the wall of the third microfluidic channel facing the foremost end . The width of the passage is measured radially to the third microfluidic channel as the distance between the foremost end and the wall of the third microfluidic channel facing the foremost end . Further, the transversal plane is oriented such that its normal facing away from the main channel has a component that is oppositely directed to a direction of flow of the further fluid in use . In this arrangement , the surface that intersects at least partially the further fluid flow forms an ef ficient trans fer zone in which or in the vicinity of which the further fluid flow is constricted in such a manner that the trans fer of emulsion microdroplets into the further fluid can take place in a reproducible manner .

[0056] In a preferred embodiment , the flow velocity of the second fluid and of the further fluid are adj usted such that the frequency of arrival of emulsion microdroplets in the trans fer zone corresponds to the formation of one double-emulsion microdroplet having a core formed by a single emulsion microdroplet .

[0057] In another embodiment , the flow velocity of the second fluid is lowered such that two emulsion microdroplets are trans ferred into the further fluid and transported at a distance from each other that allows the formation of a double-emulsion microdroplet including the two emulsion microdroplets as a core .

[0058] In a preferred embodiment , the double-emulsion microdroplets are stored at the end of the third microfluidic channel in a cooling bath containing preferably the second fluid .

[0059] In a preferred embodiment , a step of cooling at least a portion of the third microfluidic channel downstream of the passage can be provided to form a subsequent cooling area through which, in use , double-emulsion microdroplets are transported for cooling at a subsequent cooling temperature to harden . In a more preferred embodiment , the third microfluidic channel is connected fluidly downstream of the subsequent cooling area to a subsequent microfluidic channel at a subsequent j unction, protrudes in the subsequent microfluidic channel and forms a passage in the subsequent microfluidic channel . The arrangement of the channels at the subsequent j unction is similar to the arrangement of the corresponding channels at the main j unction . The subsequent microfluidic channel is designed to carry a subsequent fluid to form triple-emulsion microdroplets downstream of the subsequent j unction . The triple-emulsion microdroplets have a core formed by a doubleemulsion microdroplet surrounded by a shell made of the subsequent fluid . In addition, the microfluidic device further comprises means to control the flow velocity of the subsequent fluid . Alternatively, triple-emulsion microdroplets can be obtained by way of the microfluidic device disclosed to produce double-emulsion microdroplet . In this case , the double emulsion microdroplets are feed into the main channel while the subsequent fluid is fed into the third microfluidic channel . The same principle can be repeated to obtain further coatings around emulsion microdroplets .

[0060] In a further preferred embodiment , the microfluidic device provided is further such that it comprises means to mechanically adj ust the width of the passage , i . e . the depth of insertion of the main channel end, in the third microfluidic device in the longitudinal direction to adj ust the width of the passage , wherein the method comprises the further steps of e ) setting the width of the passage at a value that is larger, preferably 25% , more preferably 10% larger than the average diameter of the microdroplets or at a value that is smaller, preferably 30% smaller, more preferably 20% smaller than the average diameter of the microdroplets to adj ust the thickness of the further fluid surrounding the emulsion microdroplet . As already discussed above in relation to the first aspect of the invention, the width of the passage and adj usting the width of the passage has an influence on the formation of the double-emulsion microdroplets , in particular on the thickness of the shell around the core .

[0061] In a preferred embodiment , the first fluid and the further fluid are miscible . This embodiment is possible because of the hardening o f the emulsion microdroplets by cooling that takes place in a previous step . This allows a mechanical shi fting of the emulsion microdroplet in the further fluid .

[0062] In a preferred embodiment , the means to control the flow velocities , for example in the form of dispensers , comprise heating means to heat the means to control the flow to a temperature above a glass-transition temperature of the corresponding fluid, for example when the glass-transition temperature of the fluid is below room temperature . Optionally the corresponding microfluidic channels can also be heated . Preferably, the means to control the flow velocity of the first fluid and / or of the further fluid comprise heating means . Optionally, the means to control the flow velocity of the second fluid can comprise heating means .

[0063] In a preferred embodiment , the first fluid and the second fluid are chosen such that the second fluid has a viscosity at the cooling temperature that allows a flow of the second fluid to transport the emulsion microdroplets that have been cooled .

[0064] In a preferred embodiment , the first fluid and, i f present the further fluid, contains one or a combination of substances chosen in the group consisting of a thermally responsive polymer, in particular a biocompatible polymer, magnetic nanoparticles , a contrast agent , and a therapeutic agent . The contrast agent can be a micro- or nanoparticle , for example tantalum micro-particles .

[0065] In a preferred embodiment , the first fluid is a thermally responsive polymer, in particular a biocompatible polymer, containing a therapeutic agent , and the further fluid is the same thermally responsive polymer containing magnetic nanoparticles , e . g . iron oxide optionally doped with other elements , and / or a contrast agent . Magnetic nanoparticles advantageously allow guiding the emulsion microdroplets under the ef fect of an external magnetic field in a mammalian body while the contrast agent , i f present , allows the visuali zation of emulsion microdroplet by way of a visuali zation device , e . g . a X-ray apparatus , a fluoroscope , or a MRI device . The thermally responsive polymer dissolves under the ef fect of heat that can be trans ferred to the thermally responsive polymer in various manner . For example , ultrasound waves can be directed to a region where the emulsion microdroplets are located to induce a local heating . It is also possible to apply a high- frequency magnetic field by a corresponding apparatus to initiate hyperthermia to increase the temperature of the emulsion microdroplets and dissolve the polymer matrix . The dissolution triggers the release of the therapeutic agent . Separating the therapeutic agent from the nanoparticles allows for thermal shielding and drastically reduced interactions that could be detrimental to the therapeutic agent .

[0066] In a preferred embodiment , the biocompatible polymer is hydrophilic, preferably gelatin . The dissolution temperature can be tuned by altering the gelatin concentrations and / or the addition of additives , thereby influencing subsequent cross- linking . The cooling temperature at which hardening takes place can also be influenced in the same manner . Alternatively or cumulatively, additives that alter the thermal properties can be added . In a preferred embodiment , the first fluid and the further fluid contain the same thermally responsive polymer, for example gelatine , but in di f ferent concentration .

[0067] In a preferred embodiment , the second fluid is hydrophobic, preferably an oil , for example a biocompatible vegetable oil like olive oil , preferably containing a surfactant , for example sorbitan monooleate ( tradename "Span 80" ) in a concentration of 1 vol % to 5 vol% , preferably 2 vol% . Silicone oils can also be used .

[0068] In a preferred embodiment , the first fluid can be a solution of polyvinyl alcohol ( PVA) and the further fluid can be again PVA or an agar, or any other polymer or hydrogel solution .

[0069] In a third aspect of the invention, a microdroplet is disclosed that comprises a core formed by an emulsion microdroplet of a first fluid and a shell made of a further fluid, the further fluid being miscible with the first fluid, the shell being in direct contact with core , preferably the further fluid surrounding the core completely . The first fluid and the further fluid are substance that are not in the form of a gas . In a preferred embodiment , the microdroplet has a diameter of less than 2 . 0 mm, preferably less than 0 . 9 mm, more preferably less than 0 . 5 mm, most preferably less that 0 . 1 mm . Such microdroplets can be produced according to the method referring the second aspect of the invention in which a microfluidic device according to the second aspect of the invention is used .

[0070] In a preferred embodiment of the microdroplets , the first fluid and the further fluid can be chosen as described in relation to the method according to the second aspect of the invention . In a preferred embodiment , the first fluid and, i f present the further fluid, contains one or a combination of substances chosen in the group consisting of a thermally responsive polymer, in particular a biocompatible polymer, magnetic nanoparticles , a contrast agent , and a therapeutic agent .

[0071] In a preferred embodiment , the first fluid and the further fluid contain the same thermally responsive polymer, the concentration of the thermally responsive polymer in the first fluid being di f ferent to the concentration in the further fluid .

[0072] In a fourth aspect of the invention, a microfluidic device for the production of emulsion microdroplets is disclosed, the microfluidic device comprising a first microfluidic channel , a second microfluidic channel fluidly connected at a first j unction to the first microfluidic channel , and a main channel extending from the first j unction to a main channel end .

[0073] The first microfluidic channel is designed to carry a first fluid and the second microfluidic channel is designed to carry a second fluid, the second fluid being immiscible with the first fluid to develop a flow with a flow profile such that the first fluid is surrounded by the second fluid in the main channel .

[0074] The microfluidic device further comprises means to control the flow velocity of the first fluid and second fluid to allow the generation of emulsion microdroplets of the first fluid surrounded by the second f luid at the main channel end .

[0075] The microfluidic device is characteri zed by a cooling means designed to receive the emulsion microdroplets after leaving the main channel end, preferably at a free end of the main channel , and to cool them . The cooling means achieves a cooling temperature at which a hardening of the emulsion microdroplets of the first fluid takes place . Advantageously, the microfluidic device allows the formation of emulsion microdroplets that are hardened because of the cooling so that they can be stored and / or manipulated easily, for example in a further device or process .

[0076] Features related to the first microfluidic channel , the second microfluidic channel , the first j unction, and the main channel as well as the first fluid and second fluid together with the means of control their respective velocity discussed in relation to the first aspect of the invention above apply equally to the present second aspect of the invention . These features are not discussed further for this reason .

[0077] In a preferred embodiment , the main channel end is arranged in an end section of the main channel that is parallel to the direction of the gravity force . This arrangement provides for microdroplets that have a spherical shape with a small variance of their diameter .

[0078] In a more preferred embodiment , the main channel end extends in a plane perpendicular to longitudinal axis of the main channel in the end section to further improve the reproducibility of the spherical shape .

[0079] In a preferred embodiment , the cooling means is a bath containing in use the same fluid as the second fluid . Advantageously, hardened emulsion microdroplets are not contaminated by another fluid . In a fi fth aspect of the invention, a method to produce emulsion microdroplets is disclosed, comprising the steps of a ) providing a microfluidic device according to any one of the embodiments disclosed in the fourth aspect of the invention; b ) controlling the flow velocity of the first f luid and of the second fluid to generate emulsion microdroplets of the first fluid in the main channel , the second fluid being immiscible with the first fluid, and c ) generating emulsion microdroplets of the first fluid surrounded by the second fluid when leaving the main channel end for trans ferring into the cooling means , preferably a cooling bath . Leaving the main channel can be for example by j etting or dripping . d) cooling the emulsion microdroplets to harden them .

[0080] The cooling means achieves a cooling temperature at which a hardening of the emulsion microdroplets of the first fluid takes place . Advantageously, the microfluidic device allows the formation of emulsion microdroplets that are hardened because of the cooling so that they can be stored and / or manipulated easily, for example in a further device or process .

[0081] In a preferred embodiment , the cooling means is a bath containing in use the same fluid as the second fluid . Advantageously, hardened emulsion microdroplets are not contaminated by another f luid .

[0082] Preferably, the means to control the flow velocity of the first fluid and / or of the further fluid comprise are heated during the process . This can be necessary for example when the glass- transition temperature of the corresponding fluids is below room temperature . Optionally, the means to control the flow velocity of the second fluid can also be heated during the process .

[0083] The first fluid and the second fluid can be chosen in the same manner as disclosed in the second aspect of the invention .

[0084] In a preferred embodiment , the biocompatible polymer is hydrophilic, preferably gelatin . The dissolution temperature can be tuned by altering the gelatin concentrations and / or the addition of additives , thereby influencing subsequent crosslinking . The cooling temperature at which hardening takes place can also be influenced in the same manner . In a preferred embodiment , the first fluid and the further fluid contain the same thermally responsive polymer, for example gelatine , but in di f ferent concentration .

[0085] In a preferred embodiment , the second fluid is hydrophobic, preferably an oil , for example a biocompatible oil like olive oil , preferably containing a surfactant , for example sorbitan monooleate ( tradename "Span 80" ) in a concentration of 1 vol% to 5 vol% , preferably 2 vol% .

[0086] In an embodiment in which the first fluid is gelatin and the second fluid is an oil , the control of the volume of the emulsion microdroplets depends on the surface tension of the oil . However, the ratio of gelatin to oil can be modulated by altering the flow rate ratios of the first fluid and the second fluid, allowing precise tuning of capsule si zes . Description of figures

[0087] The present invention will now be described, by way of an example , with reference to the accompanying drawings in which :

[0088] Fig . 1 shows a schematic representation of the microfluidic device according to the invention and in use ;

[0089] Fig . 2 shows a schematic zoomed representation of a secondary stage of the microfluidic device to which it is referred by the reference A in Fig . 1 ;

[0090] Fig . 3 shows an experimental embodiment of the microfluidic device according to the invention;

[0091] Fig . 4 shows an experimental embodiment of the secondary stage corresponding to schematic representation of Fig . 2 ; and

[0092] Fig . 5 shows a further experimental embodiment of the secondary stage corresponding to schematic representation of Fig . 2 .

[0093] Fig . 1 represents schematically an embodiment of a microfluidic device 10 for the production of emulsion microdroplets that can be separated in a primary stage and a secondary stage , wherein the secondary stage is represented at a zoomed scale in Fig . 2 for the sake of clarity of disclosure .

[0094] The microfluidic device 10 comprises a first microfluidic channel 20 , a second microfluidic channel 30 fluidly connected and intersecting at a first j unction 40 the first microfluidic channel 20 , and a main channel 50 extending from the first j unction 40 to a main channel end 54 .

[0095] In the embodiment of Fig . 1 , the first j unction is arranged in a connection device 56 comprising a first fluid inlet 56a and a first fluid outlet 56b connected to the first fluid inlet by a connection channel 56c, wherein at least a length of the main channel 50 is inserted into the connection channel through the first fluid outlet 56b . The interface comprises further an intersection channel 56d intersecting at a right angle the connection channel 56c at an intersecting end 56e and having a second fluid inlet 56f at its end opposed to the intersecting end 56e . The first j unction 40 is formed at the intersecting end 56e .

[0096] The first microfluidic channel 20 is designed to carry a first fluid 60 and the second microfluidic channel 30 being designed to carry a second fluid 70 , the second fluid being immiscible with the first fluid .

[0097] The microfluidic device 10 can exhibit a flow transition regime in a transition region 72 of the main channel 50 after the first j unction 40 before the first fluid and the second fluid flow further and develop a laminar flow with a flow profile such that the first fluid is substantially surrounded by the second fluid in the main channel . As a result , a co- flow is created in which the first fluid and the second fluid flow in parallel in a coaxial manner through the main channel .

[0098] In the embodiment represented in Fig . 1 , the microfluidic device comprises means of control the flow velocity of the first fluid in the form of a first dispenser 80 to supply the first fluid 60 , wherein the flow rate of the first fluid is controlled by the first dispenser . Further, the microfluidic device comprises means of control the flow velocity of the second fluid in the form of a second dispenser 90 to supply the second fluid 70 , wherein the flow rate of the second fluid 70 is controlled by the second dispenser . The first dispenser and the second dispenser can be in the form of syringe pump dispensers pressuri zed by a motori zed screw for an accurate control of the flow rates .

[0099] As already known in the art , microdroplets 92 of the first fluid can be generated in the main channel by co- flow by controlling the relative flow velocities of the first fluid and second fluid . Without being exhaustive , this can occur in a dripping regime when the flow velocities of both the first and second fluid are low, wherein microdroplets of the first fluid are produced due to surface tension and set apart from the first fluid under the drag force of the second fluid . Microdroplets o f the first fluid can also be generated in a j etting regime in which the first fluid flows faster than the second fluid, causing a j et of the first fluid to expand and break up followed by the formation of spherical microdroplets of the first fluid .

[0100] The co- flow in the microfluidic device has the advantage that the first fluid is wrapped by the second fluid, thus the microdroplets are formed in a three-dimensional environment . In the present embodiment , the surface wettability of the channels , in particular the main channel , play a minor role in the formation of the emuls ion microdroplets .

[0101] The microfluidic device comprises also a cooling means 100 designed to cool at least a portion of the main channel 50 in which microdroplets 92 have been generated to form a cooling area for cooling the microdroplets . The cooling means 100 can be formed as a simple assembly comprising a cooling pad 102 and a cooling bath 104 arranged in contact over the cooling pad, wherein the main channel 50 extends in the cooling bath 104 , preferably immerged in the cooling bath to improve cooling . The length of the cooling bath 104 in the flow direction is configured such that the microdroplets are cured, i . e . hardened, when they reach the end of the portion of the main channel 50 that is cooled .

[0102] Further, in a secondary stage of the microfluidic device , the main channel 50 is fluidly connected downstream of the cooling area to a third microfluidic channel 120 at a main j unction 122 . The third microfluidic channel 120 is designed to carry a further fluid 126 , the second fluid 70 being immiscible with the further fluid 126 to develop a flow with a flow profile such that the further fluid is surrounded by the second fluid in the third microfluidic channel downstream of the main j unction 122 .

[0103] In addition, the microfluidic device 10 comprises means to control the flow velocity of the further fluid 126 to form double-emulsion microdroplets downstream of the main j unction 122 .

[0104] As illustrated in Fig . 2 , the double-emulsion microdroplets have a core formed by an emulsion microdroplet of the first fluid 60 surrounded by the further fluid 126 obtained by controlling the flow velocity of the further fluid . Means of control the flow velocity of the further fluid in the form of a further dispenser 127 is also provided for .

[0105] The hardened emulsion microdroplets allow processing in such a manner that they do not mix with the further fluid before forming double-emulsion microdroplets . The trans fer of the emulsion microdroplets into the further fluid 126 and the following formation of the shell around the emulsion microdroplets is achieved in a simple manner after the core made of the first fluid has been hardened previously in the cooling area . This allows a mechanical shi fting of the emulsion microdroplet in the further fluid .

[0106] The third microfluidic channel 120 and the main channel 50 intersect , i . e . are fluidly connected, at the main j unction 122 at an intersection angle of 90 ° .

[0107] The main channel end 54 extends in a transversal plane 128 at an angle a ( alpha ) of 50 ° with respect to a longitudinal axis 130 of the main channel , thereby forming, as seen in a longitudinal cross section of the main channel 50 , a foremost end 150 o f the main channel and an of fset end 152 of the main channel arranged upstream of the foremost end . At least a portion of the main channel end 54 intersects the third microfluidic channel and protrudes in the third microfluidic channel to form a passage 124 between the foremost end 150 and the wall of the third microfluidic channel facing the foremost end . As a result , the passage has a reduced cross-section compared to the cross-section of the third microfluidic channel . The width 154 of the passage is measured radially to the third microfluidic channel as the distance between the foremost end and the wall of the third microfluidic channel facing the foremost end 150 . Further, the transversal plane 128 is oriented such that its normal 160 facing away from the main channel has a component 162 that is oppositely directed to a direction of flow 164 of the further fluid in use .

[0108] The si ze of the angle a is directly connected to the si ze of an opening surface at the end of the main channel . The opening surface forms a surface that intersects at least partially the further fluid flow, thereby forming a trans fer zone 168 in which or in the vicinity of which the further fluid flow is constricted in such a manner that the trans fer of emulsion microdroplets into the further fluid 126 takes place .

[0109] The longitudinal axis 130 of the main channel intersects the longitudinal axis 170 of the third microfluidic channel to provide for a trans fer of the emulsion microdroplets 92 in a centered manner with respect to the direction of flow of the further fluid in the third microfluidic channel .

[0110] As a result , in use , the flow of the further fluid is reduced, as seen in cross-section, to the passage left by the main channel intersecting the third microfluidic channel . In the embodiment illustrated the width of the passage is approximately 20% smaller than the average diameter of the microdroplets . It results in a squeezing of the emulsion microdroplets and constricting the flow of further fluid .

[0111] As better seen in Fig . 2 , the main j unction 122 is arranged in an interface device 180 having a further fluid inlet 182 designed to receive a portion of the third microfluidic channel extending upstream of the main j unction and an outlet 184 designed to receive a portion of the third microfluidic channel extending downstream of the main j unction . The outlet 184 is connected to the further fluid inlet by an interface channel 186 . The interface device 180 has further a receiving channel 188 fluidly connected to the interface channel 186 and designed to receive the main channel end 54 .

[0112] Fig . 3 and Fig . 4 illustrate an experimental microfluidic device according to the invention seen from above . The microfluidic device is designed to produce double-emulsion microdroplets . The experimental microfluidic device is assembled in a similar manner as the embodiment described in relation to Fig. 1 and Fig. 2. Features present in the experimental microfluidic device having the same function as in the embodiment of Fig. 1 and Fig. 2 are referred to using the same reference numbers and are not described further. For the clarity of disclosure Fig. 4 and Fig. 5, the path followed by the microfluidic channels in the interface device has been marked in solid black lines added to the pictures.

[0113] EXAMPLE 1

[0114] In this experimental setup, a connection device 56 and an interface device 180 in the form of 3D PDMS chips (tridimensional polydimethylsiloxane chips) were utilized. The first 3D microfluidic chip acting as connection device 56 was fabricated via PDMS molding and contained a first microfluidic channel 20 with an inner diameter of 1.5 mm. The second microfluidic chip 180 acting as interface device 180 was also fabricated via PDMS molding and contained microfluidic channels with an inner diameter of 1.5mm at the receiving channel 188, outlet 184, and further fluid inlet 182, as well as an interface channel 186 with an inner diameter of 0.8 mm.

[0115] Three syringe pump modules were utilized to set the flows in combination with glass syringes of different sizes. A second dispenser 90 in the form of a 10 ml glass syringe was loaded with standardized olive oil and 2% span-80 surfactant as the second fluid 70. A first dispenser 80 and a further dispenser 127 in the form of 5 ml glass syringes were loaded with gelatine solutions of 5% for the first fluid 60 and 3% for the further fluid 126. Both gelatine-containing syringes were heated to 40 ° C using a heating mantle . In this experiment , the second dispenser 90 was maintained at room temperature .

[0116] A 16G Luer lock connector was connected to each syringe . The luer lock connector of the first dispenser 80 was directly inserted into the connection device 56 . The luer lock connector of the second dispenser 90 was connected to the intersection channel 56d using clear PVC tubing ( inner diameter 1 mm, outer diameter 2 mm) , which acted as the second microfluidic channel 30 . The same PVC tubing was used to connect the connection device 56 through the cooling bath 104 with the interface device 180 and the interface device 180 at its outlet 184 . The further dispenser 127 was connected to the interface device 180 using PTFE tubing ( inner diameter 1 . 2 mm, outer diameter 1 . 5 mm) .

[0117] During operation, monodispersed microdroplets were synthesi zed in the connection device 56 and trans ferred through the main channel 50 and the cooling bath 104 . These microdroplets underwent a sol-gel transition inside the cooling bath to form stable spherical hardened microdroplets at the main channel end 54 . Due to the geometrical constraint of the width of the passage 154 , the hardened microdroplets were forced to change from the oil phase formed by the second fluid 70 into the phase of the 3% gelatine solution formed by the further fluid 126 . The width of the passage 154 was chosen to allow for the passing of single hardened microdroplets without signi ficant pressure build-up . As a result , single microdroplets were embedded into the 3% gelatine solution forming the further fluid 126 at regular distances . In the embodiment shown, the interface channel 186 widens before the outlet 184 of the interface device 180 to improve the formation of the outer microdroplets , which were subsequently cured . Various flow rates were explored, and stabile formation of double emulsions microdroplets was observed in several flow regimes .

[0118] EXAMPLE 2

[0119] In a second experiment , the same experimental setup was utili zed to fabricate multiple emulsions microdroplets . In this experiment , the width of the passage ( 154 ) was chosen to be smaller than in example 1 , to not easily allow the passing of the hardened microdroplets . As a result , back pressure was building up and was released in regular bursts . This configuration allowed for the controlled and reproducible fabrication of multiple emulsions microdroplets .

[0120] List of reference numbers microfluidic device 10 first microfluidic channel 20 second microfluidic channel 30 first j unction 40 main channel 50 main channel end 54 connection device 56 first fluid inlet 56a first fluid outlet 56b connection channel 56c intersection channel 56d intersecting end 56e second fluid inlet 56f first fluid 60 second fluid 70 transition region of main channel 72 first dispenser 80 transition region of the main channel 82 second dispenser 90 microdroplet 92 cooling means 100 cooling pad 102 cooling bath 104 third microfluidic channel 120 main j unction 122 passage 124 further fluid 126 further dispenser 127 transversal plane 128 longitudinal axis 130 of the main channel foremost end 150 of fset end 152 width 154 angle a of transversal plane with longitudinal axis 130 normal to transversal plane 160 component of normal 162 direction of flow of the further fluid 164 trans fer zone 168 longitudinal axis 170 of the third microfluidic channel interface device 180 further fluid inlet 182 outlet 184 interface channel 186 receiving channel 188

Claims

Claims1. Microfluidic device (10) for the production of emulsion microdroplets, comprising a first microfluidic channel (20) , a second microfluidic channel (30) being fluidly connected at a first junction (40) to the first microfluidic channel (20) , a main channel (50) extending from the first junction (40) to a main channel end (54) , the first microfluidic channel (20) being designed to carry a first fluid (60) and the second microfluidic channel (30) being designed to carry a second fluid (70) , the second fluid being immiscible with the first fluid to develop a flow with a flow profile such that the first fluid is surrounded by the second fluid in the main channel , and further comprising means to control the flow velocity of the first fluid and the second fluid to allow the generation of emulsion microdroplets (92) of the first fluid in the main channel, wherein a cooling means (100) designed to cool at least a portion of the main channel (50) to form a cooling area through which, in use, emulsion microdroplets (92) are transported for cooling the emulsion microdroplets, characterized in that the main channel (50) is fluidly connected downstream of the cooling area to a third microfluidic channel (120) at a main junction (122) , wherein at least a portion of the main channel end (54) protrudes in the third microfluidic channel and forms a passage (124) between the main channel end (54) and the wall of the third microfluidic channel facing the main channel end, the width (154) of the passage being measured radially to the third microfluidic channel as the distancebetween a foremost end (150) of the main channel end (54) and the wall of the third microfluidic channel (120) facing the foremost end, the third microfluidic channel being designed to carry a further fluid (126) , the second fluid (70) being immiscible with the further fluid (126) to develop a flow with a flow profile such that the further fluid is surrounded by the second fluid in the third microfluidic channel downstream of the main junction (122) , and further comprising means to control the flow velocity of the further fluid to form doubleemulsion microdroplets downstream of the main junction (122) , the double-emulsion microdroplets having a core formed by an emulsion microdroplet of the first fluid surrounded by the further fluid.

2. Microfluidic device (10) according to claim 1, characterized in that the main channel end (54) extends in a transversal plane (128) at an angle (a) with respect to a longitudinal axis (130) of the main channel, thereby forming, as seen in a longitudinal cross section of the main channel, the foremost end (150) of the main channel and an offset end (152) of the main channel arranged upstream of the foremost end (150) , wherein at least a portion of the main channel end protrudes in the third microfluidic channel to form the passage (124) between the foremost end (150) and the wall of the third microfluidic channel facing the foremost end, the width (154) of the passage being measured radially to the third microfluidic channel as the distance between the foremost end (150) and the wall of the third microfluidic channel (120) facing the foremost end, and the transversal plane (128) is oriented such that its normal (160) facing awayfrom the main channel (50) has a component that is oppositely directed to a direction of flow (164) of the further fluid (126) in use.

3. Microfluidic device (10) according to claims 1 or 2, characterized in that the width (154) of the passage is larger, preferably 25%, more preferably 10% larger than the average diameter of the emulsion microdroplets.

4. Microfluidic device (10) according to any one of claims 1 to 3, characterized in that the width (154) of the passage is smaller, preferably 30% smaller, more preferably 20% smaller than the average diameter of the emulsion microdroplets.

5. Microfluidic device (10) according to any one of claims 1 to 4, characterized in that the main junction (122) is arranged in an interface device (180) having a further fluid inlet (182) designed to receive a portion of the third microfluidic channel (120) extending upstream of the main junction (122) and an outlet (184) designed to receive a portion of the third microfluidic channel (120) extending downstream of the main junction (122) , the outlet being connected to the further fluid inlet by an interface channel (186) , and a receiving channel (188) fluidly connected to the interface channel (186) and designed to receive the main channel end (54) .

6. Method to produce emulsion microdroplets, comprising the steps of a) providing a microfluidic device (10) according to any one of claims 1 to 5;b) controlling the flow velocity of the first fluid and of the second fluid to generate emulsion microdroplets (92) of the first fluid in the main channel (50) , the second fluid being immiscible with the first fluid, and c) cooling at least a portion of the main channel to harden the emulsion microdroplets (92) , characterized by the further steps of d) controlling the flow velocity of the further fluid, the second fluid being immiscible with the further fluid, to transfer the emulsion microdroplets from the second fluid into the further fluid and develop a flow in the third microfluidic channel (120) downstream of the passage (124) with a flow profile such that the further fluid (126) containing the emulsion microdroplets is surrounded by the second fluid (70) to allow the generation of double-emulsion microdroplets having a core formed by an emulsion microdroplet of the first fluid (60) , which core is surrounded by the further fluid (126) downstream of the passage (124) .

7. Method according to claim 6, characterized by the further steps of e) setting the width (154) of the passage (124) at a value that is larger, preferably 25%, more preferably 10% larger than the average diameter of the microdroplets or at a value that is smaller, preferably 30% smaller, more preferably 20% smaller than the average diameter of the microdroplets to adjust the thickness of the further fluid surrounding the emulsion microdroplet.

8. Method according to any one of claims 6 or 7, characterized in that the first fluid (40) and the further fluid (126) are miscible, preferably the first fluid being different to the further fluid.

9. Method according to any one of claims 6 to 8, characterized in that the first fluid and, if present the further fluid, contains one or a combination of substances chosen in the group consisting of a thermally responsive polymer, in particular a biocompatible polymer, magnetic nanoparticles, a contrast agent, and a therapeutic agent.

10. Microdroplet comprising a core formed by an emulsion microdroplet (92) of a first fluid (60) and a shell made of a further fluid (126) , the further fluid being miscible with the first fluid, the shell being in direct contact with core.

11. Microdroplet according to claim 10, characterized in that the first fluid (60) and, if present the further fluid (126) , contains one or a combination of substances chosen in the group consisting of a thermally responsive polymer, in particular a biocompatible polymer, magnetic nanoparticles, a contrast agent, and a therapeutic agent.

12. Microdroplet according to claim 11, characterized in that the first fluid (60) and the further fluid (126) contain the same thermally responsive polymer, the concentration of the thermally responsive polymer in the first fluid being different to the concentration in the further fluid.

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