[0001] This invention relates to the general technical field of assemblies for microfluidics. Aspects of the invention relate to a microfluidic attachment member, systems comprising two or more microfluidic attachment members and methods of using such members and systems for the purpose of micro mixing of fluids and droplet generation.

BACKGROUND

[0002] Microfluidics has become one of the key miniaturisation techniques to manipulate micro-, nano-, or pico-litre volumes of fluids using continuous streams of miscible fluids or segmented (droplet-based) flows of non-miscible fluids inside micrometre-sized channels. Microfluidic technology has been widely used for diagnostics, testing, screening, and materials synthesis in biotechnology and the chemical, food, pharmaceutical, and cosmetic industries (Whitesides, G.M., 2006. The origins and the future of microfluidics.

Nature, 442(7101 ), pp.368-373).

[0003]“Micro capillary devices (MCD)” which consist of coaxially assembled borosilicate glass capillaries have received a lot of attention since their invention in Weitz Lab at Harvard (Utada, A.S. et al. , 2005. Monodisperse double emulsions generated from a microcapillary device. Science,

308(5721 ), pp.537-541 ; Chu, L.-Y et al., 2010. Emulsions and Techniques for formation. U.S. Patent 7,776,927) due to their axisymmetric geometry, which allows the outer fluid to completely envelope the droplets and isolate them from the channel walls as they are formed (Takeuchi, S. et al., 2005. An axisymmetric flow-focusing microfluidic device. Advanced Materials, 17(8), pp.1067-1072).

[0004] MCDs can generate monodispersed single and multiple emulsion droplets, core-shell droplets, and micro/nano particles in a single step by using co-flow (Bandulasena, M. V. et al., 2017. Continuous synthesis of PVP stabilized biocompatible gold nanoparticles with a controlled size using a 3D glass capillary microfluidic device. Chemical Engineering Science, 171 , pp.233- 243; Othman, R. et al., 2015. Production of polymeric nanoparticles by micromixing in a co-flow microfluidic glass capillary device. Chemical Engineering Journal, 280, pp.316-329), counter current flow (Ekanem, E.E. et al. , 2015. Structured Biodegradable Polymeric Microparticles for Drug Delivery Produced Using Flow Focusing Glass Microfluidic Devices. ACS Applied Materials and Interfaces, 7(41 ), pp.23132-23143; Al nuumani, R., Bolognesi, G. & Vladisavljevicm G.T., 2018. Microfluidic production of poly(1 ,6-hexanediol diacrylate)-based polymer microspheres and bifunctional microcapsules with embedded TiO 2 nanoparticles. Langmuir, 34, pp.11822- 11831 ) or combining co-flow and counter current flow focusing (Nabavi, S.A. et al., 2017. Prediction and control of drop formation modes in microfluidic generation of double emulsions by single-step emulsification. Journal of Colloid and Interface Science, 505, pp.315-324; Nabavi, S.A. et al., 2015. Double emulsion production in glass capillary microfluidic device: Parametric investigation of droplet generation behaviour. Chemical Engineering Science, 130, pp.183-196; Utada, A.S. et al., 2005. Monodisperse double emulsions generated from a microcapillary device. Science, 308(5721 ), pp.537-541 ) patterns.

[0005] Traditional glass capillary devices are made up by bonding coaxial capillaries onto a microscope slide and fluids are supplied via syringe needles attached to the capillary ends. The fabrication process of these devices is tedious, labour intensive and time consuming because the capillaries are manually aligned. Sometimes, several devices are wasted before a satisfactory coaxial alignment of the capillaries is achieved. In addition, the device is difficult to clean or sterilise, since the capillaries cannot easily be separated from the glass slide. Besides, the alignment of the capillaries may be lost because the epoxy glue softens at high temperatures.

[0006] Alternative methods to fabricate microfluidic devices such as etching techniques, hot embossing, and laser ablation are more expensive than capillary pulling and polishing (Vladisavljevic, G.T., Kobayashi, I. & Nakajima, M., 2012. Production of uniform droplets using membrane, microchannel and microfluidic emulsification devices. Microfluidics and Nanofluidics, 13(1 ), pp.151 -178). Soft lithography is a well-established method for rapid prototyping of microfluidic devices in polydimethylsiloxane (PDMS). However, PDMS is a hydrophobic polymer and inconvenient for making oil-in-water (O/W) emulsions (Fiorini, G.S. & Chiu, D.T., 2005. Disposable microfluidic devices: Fabrication, function, and application. BioTechniques, 38(3), pp.429-446). In addition, organic solvents can interact with PDMS and cause its swelling, extraction of un-polymerized monomers/oligomers from the polymer network, permeation of hydrophobic solutes into PDMS walls, and chemical decomposition of PDMS (Fiorini, G.S. & Chiu, D.T., 2005. Disposable microfluidic devices: Fabrication, function, and application. BioTechniques, 38(3), pp.429-446; Lee, J.N., Park, C. & Whitesides, G.M., 2003. Solvent Compatibility of poly(dimethylsiloxane)-based microfluidic devices. Analytical Chemistry, 75(23), pp.6544-6554). Swelling can significantly alter the dimensions of the PDMS channels and may even cause a complete channel closure (Van Dam, R.M., 2006. Solvent-Resistant elastomeric microfluidic devices and applications. California Institute of technology). Extraction of non-crosslinked species can introduce contaminants into different process streams and cause product contamination. Partitioning of solutes between fluid streams and cured PDMS can alter concentrations of reagents and trapped solutes can be released subsequently from the channel walls.

[0007] PDMS channels are also difficult to clean because PDMS chips are permanently bonded to glass slides. In traditional glass capillary devices developed by Weitz lab (Utada, A.S., et al., 2005. Monodisperse double emulsions generated from a microcapillary device. Science, 308(5721 ), pp.537-541 ), capillary tubes and needles for fluid delivery are sealed to the glass slide using epoxy glue.

[0008] As described above, traditional glass capillary devices are time consuming to fabricate and are not reusable. Due to the disposable nature of the devices, reproducibility is one of the main issues inherent to glass capillary microfluidic devices.

[0009] Aspects and embodiments of the present invention seek to address and mitigate problems associated with the prior art. BRIEF SUMMARY OF THE DISCLOSURE

[0010] According to the present invention, there is provided a microfluidic attachment member, comprising: a channel configured to support a capillary tube and configured to be removably coupled to a further attachment member to assemble a microfluidic module, wherein when the attachment member is coupled to another attachment member, the channels co-axially align so that they can hold an outer capillary tube.

[0011] Another aspect of the present invention, there is provided a system comprising two or more of the microfluidic attachment members described herein.

[0012] In another aspect of the present invention, there is provided a method of micro-mixing fluids or droplet generation using the system described herein comprising supplying two or more fluids to at least one of the microfluidic attachment members and collecting the micro-mixed fluid or generated droplets from at least one of the microfluidic attachment members.

DESCRIPTION OF THE DRAWINGS

[0013] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0014] Figure 1A shows a plan view of the attachment member (10) comprising a channel (13) to hold the outer capillary tube (19), a channel (15) to hold the inner capillary tube (20 or 20’), fluid port (14) to deliver a fluid to the annular region between the inner and outer capillary tubes, port (16) to house a standard tube connector (22) which immobilised the inner capillary tube (20 or 20’) and the stud-and-tube coupling system composed of a stud (17) and a tube (18).

[0015] Figure 1 B shows an isometric view of the attachment member (10).

[0016] Figure 2A shows a plan view of the attachment member (11 ) comprising an extended channel (13’) to hold the outer capillary tube (19’) and the stud-and- tube coupling system composed of a stud (17) and a tube (18).

[0017] Figure 2B shows an isometric view of the attachment member (11 ). [0018] Figure 3A shows a plan view of the attachment member (12) comprising a channel (13) to hold the outer capillary tube (19), fluid port (14) to deliver a fluid to the outer capillary tube (19) and the stud-and-tube coupling system composed of a stud (17) and a tube (18).

[0019] Figure 3B shows an isometric view of the attachment member (12).

[0020] Figure 4A is a photograph of two attachment members (10) joined together to make the microfluidic module (1 ). The coaxial assembly of capillaries comprises an outer capillary tube (19), an inner injection capillary tube (20) and an inner collection capillary tube (20’). There are four tube connectors (22); two of which are attached to medical tubing (21 ) and the other two capable of holding inner capillaries (20 or 20’) securely inside ports (16).

[0021] Figure 4B shows a plan view of the microfluidic module (1 ) obtained by assembling two attachment members (10) using the stud and tube coupling system and inserting the capillary tubes. The channel (13) is capable of holding the outer capillary tube (19). Channels (15) are capable of holding two inner capillary tubes (20 or 20’) at each side of the module generating the combination of co-flow and counter current flow geometry. The system comprises two fluid ports (14a & 14b) to deliver two different fluids to the annular region between the inner and outer capillary tubes at each side of the module and two ports (16a & 16b) to house standard tube connectors (22) which immobilise the inner capillary tubes (20 or 20’).

[0022] Figure 5 shows a plan view of the microfluidic module (2) obtained by joining together one attachment member (10), and one attachment member (11 ) using the stud and tube coupling system and inserting the capillary tubes (19’ and 20). The channels (13 & 13’) are capable of holding the outer capillary tube (19’) and channel (15) is capable of holding the inner injection capillary tube (20) generating the co-flow geometry. The system also comprises of a fluid port (14) to deliver a fluid to the annular region between the inner injection capillary tube and the outer capillary tube and port (16) to house a standard tube connector (22) which immobilises the inner injection capillary tube (20).

[0023] Figure 6 shows a plan view of the microfluidic module (3) obtained by joining one attachment member (10), and one attachment member (12) using the stud and tube coupling system and inserting the capillary tubes. The channels (13) are capable of holding the outer capillary tube (19) and channel (15) is capable of holding the inner collection capillary tube (20’) generating the counter current flow focusing geometry. The system also comprises of two fluid ports (14a & 14b) to deliver fluids to the annular region between the inner and outer capillary tubes and inside the outer capillary tube, respectively, as well as port (16) to house a standard tube connector (22) which immobilised the inner collection capillary tube (20’).

[0024] Figure 7 shows photographs of three different capillary devices assembled by interlocking different attachment members and creating different flow geometries inside the microfluidic modules. B1 block is equivalent to attachment member 10. B2 is equivalent to attachment member 11. B3 is equivalent to attachment member 12.

[0025] Figure 8 shows the flow patterns during liposome formation at different organic and water stream flow rates in the co-flow geometry.

[0026] Figure 9 shows the UV-Vis absorbance spectra of AuNPs synthesised using variable flow rates of ascorbic acid and the effect of ascorbic acid flow rate on the size of synthesised AuNPs whilst keeping the FIAuCU flow rate constant.

[0027] Figures 10 and 11 show the formation of W/O single emulsion droplets in the counter-current flow focusing geometry.

[0028] Figure 12 shows the formation of W/O/W double emulsion droplets in co-flow and counter current flow combination geometry.

[0029] Figure 13 shows the fluorescent images of the single core, double core and triple core double emulsion droplets collected using the W/O/W emulsion droplet generation geometry.

[0030] Figure 14 demonstrates the option to change the inner capillary positions in order to manipulate the droplet generation.

[0031] Figure 15 shows the micro-mixing of reagents to synthesise AgNPs using 3D droplets in 3 phase droplet generation geometry.

DETAILED DESCRIPTION

[0032] A microfluidic attachment member, comprising: a channel configured to support a capillary tube and configured to be removably coupled to a further attachment member to assemble a microfluidic module, wherein when the attachment member is coupled to another attachment member, the channels co-axially align so that they can hold an outer capillary tube.

[0033] Preferably, wherein the channel extends through the attachment member from a first opening through to a second opening. Advantageously, having the channel extend through the attachment member such that it has two openings allows for many additional uses. Capillary tubes may be inserted from either ends, increasing the flexibility of cleaning and experimental or operating procedures. Cleaning is made easier since a cleaning fluid could enter a first opening and exit through the second opening.

[0034] In an aspect, the attachment member further comprises at least one fluid port located at an outer face of the module. Preferably, wherein the at least one fluid port and the capillary holder channel are in fluid connection via a connection channel formed between the fluid port and the channel within the attachment member.

[0035] Advantageously, each attachment member can be configured differently to allow for alternate combinations of the attachment members to produce different flow patterns such as co-flow, counter current flow or combination of co-flow counter current flow when the attachment members are used as part of a microfluidic module.

[0036] In use, each attachment member can be assembled with a further attachment member which when combined can form a microfluidic module for microfluidic experimentation. The outer capillary holder channel in each of the attachment member can house an outer capillary tube (borosilicate glass capillary tubes are preferred) which is held in place between the attachment members, allowing the user to have a top or bottom view of the outer capillary tube, making it clearly visible to an inverted or standard optical microscope. Importantly, fluid flow rates, pressures and positioning of inner capillaries can only be fine-tuned if the boundary between the different fluids and/or the position of inner capillaries are clearly visible. Inverted optical microscopes are common in the field of microfluidic and require transparent or translucent substrates to function. [0037] In a further embodiment of the present invention, the outer capillary holder channel and the inner capillary holder channel are coaxial with the inner capillary holder port. Having the capillary holder channels coaxial with the inner capillary holder port allows self-alignment of the inner capillary with the outer capillary.

[0038] In a further embodiment of the present invention, an attachment member comprises a male protrusion and a female alignment hole configured to releasably couple to a female alignment hole and a male protrusion of a further attachment member to form a microfluidic attachment member. Coupling the attachment members ensures that the capillary tubes will not move around during experiments since the capillary holding channels and capillary holding ports are holding the capillary tubes securely. The attachment member(s) are coupled using the stud and hole coupling system such that no significant movement of the attachment members relative to each other is possible and to hold the microfluidic attachment member together such that the forces produced by fluid pressure or a user knocking the assembly will not break the assembly. As an extra precaution, a customised stage can be used to hold the fully assembled microfluidic attachment member.

[0039] Utilising an attachment member comprising a male protrusion configured to releasably attach to a female alignment hole of another attachment member will allow for easy coupling and decoupling of the attachment members to each other. If a stud and a tube are present on each of the connected attachment member then precise axisymmetry of the capillaries will be achievable. This allows for greater ease of coaxially aligning inner capillary tubes with outer capillary tubes.

[0040] Optionally, wherein the attachment member is a snap-fit attachment member.

The coupling system also be a stud-and-tube coupling system which allows the attachment members to be interchangeable.

[0041] Preferably, the attachment members are formed of a chemically inert polymer, preferably wherein the polymer is a polyacetal (polyoxymethylene) copolymer or cyclic olefin copolymer. The microfluidic attachment member may have a flat shape which allows improved stacking when there is more than one module. The microfluidic attachment members can be stacked vertically, or arranged horizontally. There is no limit to the number of members which may be connected or stacked.

[0042] Advantageously, the attachment members of the present invention can be reusable, can be made of cheap material and can be inexpensive to manufacture. Three different attachment member blocks were designed using SolidWorks software (Dassault Systemes), a solid modelling computer-aided design (CAD) and computer-aided engineering (CAE) computer program and manufactured using a fully automated CNC milling machine (HAAS Automation, model Super Mini, Norwich, UK).

[0043] In a further embodiment there is provided a system comprising more than two attachment members of the present invention.

[0044] The system comprises an outer capillary tube supported in the channels of the first and further attachment members.

[0045] Preferably, the system further comprises at least one inner capillary tube located at least partially within the outer capillary tube providing fully axisymmetric round-capillary-inside-round-capillary geometry which provides 100% uniform flow 360° around the inner capillary orifices compared to that of the round capillary inside a square capillary arrangement which generates unnecessary vortices due to the unevenness of the cavity around the inner capillary orifices.

[0046] According to one aspect, at least one of the attachment members comprises at least one fluid port located at an outer face of the microfluidic module, preferably wherein the system comprises at least one entry fluid port and at least one collection port.

[0047] Optionally, wherein the at least one inner capillary tube is in fluid contact with one of the at least one fluid ports, optionally wherein the at least one inner capillary tube comprises a tapered orifice. The orifice may be a nozzle.

[0048] The fluid port can house a standard tube connector such that fluids can be introduced and flow to the capillary holder channel which holds the outer capillary tube. This allows for the pumping/delivery of fluids into the outer capillary tube, if two of the attachment members have a port each then at least two separate fluids can be input from each side, which allows for generating two-phase micro-mixing or the formation of droplets with the outer capillary being used as the outer boundary of the microfluidic geometry. This allows for experiments wherein a two-fluid interface is required, such as creating emulsion droplets or experiments to synthesise precisely sized nanoparticles wherein the resulting size is controlled by the flow rate of two or more fluid streams.

[0049] If two of the attachment members have two fluid ports then three separate fluids can be input, which allows for generating three-phase micro-mixing or core-shelled droplets with the fourth port acting as an outlet port into which the mixed fluid or core-shelled droplets can flow out. In practice a fluid tight seal will be formed between an inner/outer capillary tubes and the capillary holder channels and ports.

[0050] In accordance with the present invention there is provided a method of micro mixing fluids using the system comprising microfluidic modules in accordance with the invention described herein. The method comprises supplying two or more fluids to the microfluidic module and collecting the micro-mixed fluid from the microfluidic module.

[0051] Preferably wherein the attachment members of the microfluidic modules can be combined in alternate ways to achieve different micro-mixing geometries.

[0052] Advantageously, these microfluidic modules are used to form:

(i) single emulsion droplets;

(ii) multiple emulsion droplets;

(iii) core-shell droplets and encapsulated droplets;

(iv) micro-mixing of continuous streams;

(v) micro-mixing within droplets;

[0053] Advantageously, the diameter of the droplets and/or shell thickness can be altered by adjusting the distance between the inner capillary orifices.

[0054] Having an inner capillary tube which is located within the outer capillary tube facilitates experiments and methods of production wherein multiple fluids need to converge in the same location. [0055] A further aspect of the present invention provides a method of coupling a first and a further attachment member, comprising the steps of: i) providing a first attachment member, comprising: an outer capillary holder channel configured to support a outer capillary tube to be coupled via the stud and tube coupling system to a second attachment member, the outer capillary holder channel of the first attachment member to coaxially align with the outer capillary holder channel of the second attachment member and an outer capillary tube to be mounted within both the channels and both the attachment members; ii) providing a first attachment member comprising: an inner capillary holder channel configured to support an inner capillary tube, to be coupled via the stud and tube coupling system to a second attachment member with an inner capillary holder channel, the channel of the first attachment member to coaxially align with the inner capillary holder channel of the second attachment member and an inner capillary tube to be mounted within all the inner capillary holder channels and outer capillary holder channels; iii) inserting the outer capillary tube into the outer capillary holder channel of the first attachment member; iv) coupling the first attachment member to the second attachment member by using the stud and tube coupling system and inserting the outer capillary tube into the outer capillary holder channel of the second attachment member.

[0056] Advantageously the provided method of coupling two attachment members does not require any gluing and is non-permanent meaning the capillary tubes and attachment member can be replaced, unattached and cleaned with ease.

[0057] A attachment member can further comprise a fluid port located at an outer face of the attachment member, wherein the fluid port and the capillary holder channels are in fluid connection via a connection channel formed between the fluid port and the capillary holder channel within the attachment member, and further comprising the step of:

[0058] (v) inserting a one fluid standard connector into the fluid port of the attachment member. [0059] In a further embodiment an attachment member can comprise an inner capillary holder port located at the end of the attachment member, wherein the inner capillary tube will be inserted.

[0060] (vi) inserting an inner glass capillary tube with standard tube connectors into the inner capillary holder port of the attachment member.

[0061] List of reference numerals

1 - microfluidic module comprising two (10) attachment members

2 - microfluidic module comprising one (10) attachment member and 1 (11 ) attachment member

3 - microfluidic module comprising one (10) attachment member and 1 (12) attachment member

10 - attachment member with one fluid port and one capillary holder port

11 - attachment member with one capillary holder channel

12 - attachment member with one fluid port

13 - outer capillary holder channel

13’ - extended channel

14 - fluid port

15 - inner capillary holder channel

15’ - connection channel

16 - port

17 - male protrusion

18 - female alignment hole

19 - outer capillary tube

19’ - extended outer capillary tube

20 - inner (injection) capillary tube

20’ - inner (collection) capillary tube

21- medical tubing

22 - standard tube connector 23 - customised microfluidic module holder stage

24 - observation window

[0062] In this specification, the following terms may be understood in view of the below explanations:

[0063] The term“fluid port” may refer to a port into which a standard tube connector may be releasably connected.

[0064] The term“port” may refer to a port into which a standard tube connector releasably inserted with an inner capillary.

[0065] The term“outer capillary holder channel” may refer to a channel into which the outer capillary tube inserted.

[0066] The term “connection channel” may refer to any closed or open channel through which a fluid may flow. Preferably, the connection channel may be any diameter wherein the fluid flow becomes laminar rather than turbulent. Generally, the diameter of the channel will be within the range of 1 100 micrometres to 2000 micrometres.

[0067] The term “fluid contact” may refer to a situation in which two or more components or features are joined such that a fluid may flow between them.

[0068] The term“tapered” may refer to any gradual reduction in radial diameter along an axial length.

[0069] The term“snap-fit” may refer to a component which has features that can interlock with the features of another component. The features can then be held in contact by hoop or torsional strain or a lever/ pin-based mechanism or similar means.

[0070] The term“fluid” may refer to any matter which can flow including both liquids and gases.

[0071] Figures 1A and 1 B show an attachment member (10) having a rectangular parallelepiped shape with a rectangular cavity in the middle comprising a channel (13) for holding and positioning the outer capillary tube (19), a channel (15) for holding and positioning the inner capillary tube (20) or (20’) which is shown in Figure 4B, one fluid port (14) which is perpendicular to the channel (15). The fluid port (14) which has a threaded internal structure for the attachment of standard tube connectors with matching threads is in fluid contact with the channel (15) via connection channel (15’) which has a smaller diameter than the fluid port. The attachment member also comprises of a port (16) with threaded internal structure for the attachment of standard tube connectors with matching threads to immobilise inner capillary tubes inside the inner capillary holder channel (15). The attachment member (10) further comprises of a male protrusion (17) and a complimentary female alignment hole (18) to facilitate the attachment with another attachment member.

[0072] Figures 2A and 2B show an attachment member (11 ) having a rectangular parallelepiped shape with a rectangular cavity in the middle comprising a channel (13’) for holding an outer capillary tube (19’) which extends through the body of the attachment member such that it has two openings at either ends. The attachment member (11 ) further comprises of a male protrusion (17) and a complimentary female alignment hole (18) to facilitate the attachment with a further attachment member.

[0073] Figures 3A and 3B show an attachment member (12) having a rectangular parallelepiped shape with a rectangular cavity in the middle comprising a channel (13) for holding an outer capillary tube (19) and a fluid port (14) located perpendicular to the channel (13). The fluid port (14) which has a threaded internal structure for the attachment of standard tube connectors with matching threads is in fluid contact with the channel (13) via connection channel (15’) which has a smaller diameter than the fluid port. The attachment member also comprises of a port (16) with a threaded internal structure for the attachment of standard tube connectors with matching threads to immobilise inner capillary tubes inside the inner capillary holder channel (15). The attachment member further comprises of a male protrusion (17) and a complimentary female alignment hole (18) to facilitate the attachment with an additional attachment member creating a rectangular observation window (24).

[0074] Figure 4A shows a photograph of the microfluidic module (1 ) which is assembled by connecting two attachment members (10) using respective male protrusions and complimentary female alignment holes and create the observation window (24) between them. Both attachment members hold an outer capillary tube (19) between them in their respective channels (13) which can be seen through the observation window as shown in Figure 4B. The two fluid ports hold medical tubing (21 ) connected to them via standard tube connectors (22) which allow for fluids to be fed from an external source container into the microfluidic module.

[0075] Figure 4B shows a schematic of the microfluidic module (1 ) which is assembled by connecting two attachment members (10) using respective male protrusions (17) and complimentary female alignment holes (18) and create the observation window (24) between them. An outer capillary tube (19) is held by the channels (13) at the opposite internal faces of the observation window (24) of the microfluidic attachment member. There are two fluid ports (14a and 14b) which can be connected to medical tubing (21 ) using standard tube connectors (22). There are two ports (16a and 16b) which are used to hold and immobilise the inner capillary tubes (20 or 20’) using standard tube connectors (22). In the present example the port (16a) houses the inner injection capillary tube (20) which has a smaller diameter than the outer capillary tube (19) which may inject a first fluid directly into the outer capillary tube (19). The port (16b) houses the inner collection capillary tube (20’) of the same diameter as the inner injection capillary tube (20) which is located proximal to the orifice of the inner injection capillary tube (20) and allows the product to flow out of the microfluidic attachment member. The fluid ports (14a and 14b) house medical tubing (21 ) using standard tube connectors (22) allowing for fluids to enter the microfluidic attachment member. In this embodiment the inner injection capillary tube (20) and the inner collection capillary tube (20’) are tapered and orifices are made to required diameters.

[0076] Figure 5 shows a schematic of the microfluidic module (2) which is assembled by joining an attachment member (10) to an attachment member (11 ) using respective male protrusions (17) and complimentary female alignment holes (18) and create the observation window (24) between them. An outer capillary tube (19’) is held in the channels (13 and 13’) of the two attachment members (10 and 11 ). The attachment member (11 ) has a channel (13’) with two openings such that an extended capillary tube (19’) can fit into the two channels (13 and 13’). In the present example, there is one fluid port (14) which is connected to medical tubing (21 ) using standard tube connectors (22). The port (16) houses a standard tube connector (22) which facilitate the inner injection capillary tube (20) to be immobilised to the microfluidic attachment member (2). In this embodiment the outer capillary tube (19’) extends beyond the attachment member (11 ) to collect the products from the microfluidic module (2) or to send it to further processing steps downstream. The inner injection capillary tube (20) is tapered to make the orifice diameter smaller than its inner diameter.

[0077] Figure 6 shows a schematic of the microfluidic module (3) which is assembled by joining an attachment member (10) and an attachment member (12) using respective male protrusions (17) and complimentary female alignment holes (18) and create the observation window (24) between them. An outer capillary tube (19) is held in the channels (13) of the two attachment members. There are two fluid ports (14a and 14b) each of which are connected to medical tubing (21 ) using standard tube connectors (22). There is also a port (16) which houses a standard tube connector (22) to immobilise the inner collection capillary tube (20) in the microfluidic module (2).

[0078] Figure 7 shows photographs of three different capillary devices assembled by interlocking different attachment members: (a) Two-phase co-flow; (b) Two- phase counter-current flow; (c) Three-phase flow. The schematics of flow patterns inside the devices are also shown. Interaction between miscible liquid streams leads to micro-mixing (i), while interaction between immiscible liquid streams leads to drop generation (ii). Micro-mixing and drop generation can be combined in same device (Figure ci)

[0079] Figure 8 shows flow patterns during liposome formation at different organic and water stream flow rates: (a) Q ₀ = 0.6 ml/h, Q _w = 0.6 ml/h, D _v = 557 nm, PDI = 0.245; (b) Q ₀ = 0.6 ml/h, Q _w = 5 ml/h, D _t = 675 nm, PDI = 0.283; (c) Q ₀ = 12 ml/h, Q _w = 25 ml/h, D _v = 262 nm, PDI = 0.145; (d) Q ₀ = 20 ml/h, Q _w = 25 ml/h, D _v = 222 nm, PDI = 0.093, D _t = 260 pm.

[0080] Figure 9 shows the effect of variable flow rate of ascorbic acid stream on the absorbance spectra, the synthesised AuNPs, and the wavelength of maximum absorbance at the orifice diameter of Di= 100 m and the constant flow rate of HAuCU stream of 15 ml/h.

[0081] Figure 10 shows the formation of W/O emulsions in counter-current flow focusing geometry with Di= 180 pm: (a) Q _d = 1.9 ml/h, Q _c = 9 ml/h; (b) Q _d = 3 ml/h, Q _c = 7 ml/h with the corresponding droplet diameter of: (c) 80.6 ± 1.3 pm at Q _c /Q _d = 4.7; (d) 122.8 ± 2.9 pm at Q _c /Q _d = 2.3.

[0082] Figure 11 shows the generation of W/O emulsion over 6 hours by counter- current flow focusing using glass capillary device with Di= 200 pm at Q _c = 15 ml/h and Q _d = 1.5 ml/h: (a) t = 0 h; (b) t = 2 h; (c) t = 4 h; (d) t = 6 h. The average droplet diameter is 104.5 ± 5.1 pm.

[0083] Figure 12 shows single and multiple core W/O/W emulsion droplets generated in a three-phase device with D _t = 50 pm and D _c = 300 pm: (a) Q _ip = 4 ml/h, Qm _P = 6 ml/h, Q _op = 25 ml/h, quadruple core encapsulation; (b) Q _ip = 2.5 ml/h, Qm _P = 6 ml/h, Q _op = 25 ml/h, double core encapsulation; (c) Q _ip = 2 ml/h, Q _mp = 6 ml/h, Q _op = 25 ml/h, single core encapsulation; (d) Q _ip = 2 ml/h, Q _mp = 3 ml/h, Q _op = 20 ml/h, single core encapsulation with thinner shells (e), (f), (g), and (h) are the micrographs of generated monodispersed droplets. Eccentric position of some cores was due to difference in density between the core and shell fluid.

[0084] Figure 13 shows W/O/W emulsion droplets with Calcein and Nile red encapsulated in the core and shell fluid: (a) core-shell droplets with the core diameter of 258 ± 4 pm and the shell diameter of 391 ± 7 pm; (b) double core multiple emulsion droplets with the core diameter of 240 ± 8 pm and the shell diameter of 503 ± 9 pm; (c) triple core multiple emulsion droplets with the core diameter of 251 ± 3 pm and the shell diameter of 550 ± 8 pm.

[0085] Figure 14 shows the effect of the distance I between injection and collection capillary orifice on the morphology of W/O/W emulsion droplets formed at Q _ip = 2 ml/h, Q _mp = 6 ml/h, Q _op = 25 ml/h, D _t = 50 urn, and D _c = 300 pm.

[0086] Figure 15 shows the preparation of AgNPs within droplets by mixing 3 mM aqueous AgN0 ₃ solution and 35 mM aqueous tannic acid solution at pH = 8. The flow rate of Miglyol 840 varied from 15 to 20 to 25 ml/h, while the flow rates of AgNC and tannic acid streams were fixed at 2 ml/h and 6 ml/h, respectively.

EXPERIMENTAL

Chemicals

[0087] Preparation of nanovesicles and nanoparticles. Lipoid ^® E80 (Lipoid GmbH, Ludwigshafen, Germany, egg yolk lecithin containing 82% phosphatidylcholine and 9% phosphatidylethanolamine) and cholesterol (Sigma-Aldrich Chemicals, Saint Quentin Fallavier, France) were used to form liposomes. Lipids were dissolved in analytical grade ethanol (Fisher Scientific, UK). Milli-Q water prepared using the Millipore 185 Milli-Q Plus unit was used as anti-solvent. Hydrogen tetrachloroaurate(lll) (HAuCU, ³99.9% trace metals basis, Sigma-Aldrich, UK) and polyvinylpyrrolidone (PVP K30, M _w ~40,000 g/mol, Sigma Aldrich, UK) were used as gold precursor and capping agent for gold nanoparticles (AuNPs), respectively. A reagent grade L-ascorbic acid (Sigma-Aldrich, UK) was used as a reducing agent. The pH of the ascorbic acid solution was adjusted with 2 M NaOH supplied by Fisher Scientific, UK. Silver nanoparticles (AgNPs) were prepared by mixing AgN0 ₃ and tannic acid, both supplied by Sigma-Aldrich.

[0088] Preparation of emulsions. In W/O/W emulsions, glycerol (Fisher Scientific, UK) was used to modify viscosity, XIAMETER ^® RSN-0749 resin (a 50/50 mixture of trimethylsiloxysilicate and cyclomethicone, Univar, UK) and poly(vinyl alcohol) (PVA, M _w = 13,000-23,000 g/mol, 87-89% hydrolysed, Sigma-Aldrich, UK) were used as lipophilic and hydrophilic surfactant, respectively. The oil phase was Dow Corning ^® 200, 10 cSt fluid (VWR, UK). Nile red and Calcein (Sigma-Aldrich) were used for fluorescent labelling. The carrier oil in W/O emulsions was Miglyol ^® 840 (Sasol, Germany), a propane- 1 ,2-diol diester of caprylic acid (65-80%), capric acid (20-35%), caproic acid (<2%), lauric acid (<2%), and myristic acid (<1 %). In some cases, Eudragit S100 (Evonik, Germany), a pH sensitive copolymer of methacrylic acid and methyl methacrylate (1 :2) was dissolved in the aqueous phase and 4- aminobenzoic acid (99%, Acros Organics, UK) was added in the oil phase as a crosslinker. Experimental Equipment

Capillaries

[0089] Borosilicate round capillary tubes with 2.0 mm OD / 1.56 mm ID and 1.0 mm OD / 0.58 mm ID (World Precision Instruments, UK) were used as inner and outer capillaries, respectively. Inner capillaries were pulled using a P-97 micropipette puller (Sutter Instrument Company, USA) and pulled tips were adjusted to the desired orifice size by grazing against abrasive paper. The orifice size was measured using a Narishige MF-830 microforge (Linton

Instrumentation, UK). The capillaries were treated with octadecyltrimethoxysilane and 2-

[methoxy(polyethyleneoxy)propyl]trimethoxysilane to render the surface hydrophobic and hydrophilic, respectively.

Microfluidic modules

[0090] Three different attachment members were designed using SolidWorks

software (Dassault Systemes), a solid modelling computer-aided design (CAD) and computer-aided engineering (CAE) computer program and manufactured using a fully automated CNC milling machine (HAAS

Automation, model Super Mini, Norwich, UK). The material used for fabrication was polyacetal (polyoxymethy!ene) copolymer with a density of 1.41 g/cm ³

Experimental set-up

[0091] . Fluids were delivered using 11 Elite syringe pumps (Harvard Apparatus, UK) from SGE gas-tight glass syringes (10 ml, 25 ml, and 50 ml, Sigma-Aldrich, UK) via fine-bore Portex polyethylene medical tubing (1.52 mm OD / 0.86 mm ID, Smiths Medicals, UK), and Omnifit ^® or Super Flangeless™ tube connectors. The device was mounted on the stage of an inverted biological microscope (GXM-XDS-3, GT Vision, UK), Droplet generation and mixing process were observed and recorded using a Phantom V5.1 high speed camera with a resolution of 576 ^c 576 pixels. ImageJ program was used to process the video recordings and determine the droplet diameters. Data Analysis

[0092] The size, D _v and polydispersity index, PDI of liposomes and silver nanoparticles were measured by photon correlation spectroscopy using a Delsa™ Nano HC particle analyser (Beckman Coulter, UK). The absorption spectra of synthesised AuNPs were recorded using a Perkin Elmer Lambda 35 UV/VIS spectrometer.

Flow patterns in the Device

[0093] Three devices were assembled by combining different blocks and corresponding flow patterns in the device. Using different combinations of the microfluidic modules and miscible or immiscible liquid streams, the device can be operated as a co-flow, counter-current flow focusing or three-phase micromixer or droplet generator.

Results and Discussion

Two-phase co-flow

[0094] Preparation of liposomes: An ethanolic solution of 20 mg/ml Lipoid ^® E80 and 5 mg/ml cholesterol was injected through the inner capillary, while Milli-Q water was introduced through the annular space between the inner and outer capillary. After injection, ethanol and lipids diffuse from the core region into water in the annular region, while water molecules diffuse in the opposite direction. When the solubility limit of the lipids is exceeded, they self- assemble into disk-like bilayer structures, which tend to curve upon growing, and eventually close to form a spherical vesicle. Under mild shear forces and/or at high concentration, vesicles tend to aggregate into clusters. This vesicle aggregation is reversible, but bilayer defects may induce irreversible fusion of bilayers in clusters.

[0095] At low flow rates, two streams flow side by side with a sharp liquid boundary, heavy vesicle aggregation in the aqueous phase and a high ethanol concentration in the core region. The mixing time of lipids dissolved in the organic stream is: t _mix ~ D?/D, where D _t is the orifice diameter of the injection tube and D = 0.84x1 O ⁹ m ² /s is the diffusion coefficient of ethanol in water (Cussler, E.L., 2009. Diffusion: Mass Transfer in Fluid Systems, 3 ^rd Edition, Cambridge University Press, Cambridge, p. 127.). The residence time of the organic stream in the device is: t _res - LDf /Q ₀ , where L -9 cm is the length of the collection capillary downstream of the injection point. For efficient mixing, t _res > t _mix or /D* > Pe, where Pe is the Peclet number. In Figure 7(a), L/Di -350 and Pe -760, which means that t _res > t _mix , i.e. a complete mixing cannot be achieved in the device. Due to high Q _w /Q ₀ value, high concentration gradients were established at the interface, which led to the build-up of transient interfacial tension between ethanol and water (Lacaze, L., Guenoun, P., Beysens, D., Delsanti, M., Petitjeans, P., Kurowsk, P., 2010. Transient surface tension in miscible liquids. Physical Review E, 82, 041606). As a result, the interface around the injection nozzle acquired a hemispherical shape typical for dripping regime with immiscible liquids. The fusion of bilayers due to heavy vesicle aggregation led to large vesicle sizes of 550- 680 nm.

[0096] When fluid flow rates were relatively high, the interface was extended into a widening jet and a vortex flow was formed around the jet interface. When lipid bilayer fragments (LBFs) bend from a flat disc into a closed sphere, the energy of a LBF first increases due to bending contribution and then decreases as the edges of the disc meet and disappear. Energy dissipation of the vortex ring can help to achieve bending of the flat bilayer discs at smaller diameters resulting in smaller vesicles with lower PDI, which agrees with previous study (Vladisavljevic, G.T., Laouini, A., Charcosset, C., Fessi, H., Bandulasena, H.C.H., Holdich, R.G., 2014. Production of liposomes using microengineered membrane and co-flow microfluidic device. Colloids Surfaces A Physicochem. Eng. Asp. 458, 168-177).

[0097] Synthesis of AuNPs: Synthesis of AuNPs was carried out by injecting 1 mM FIAuCU solution containing 1 % (w/v) PVP K30 through the injection tube with 100 pm orifice into 20 mM aqueous solution of ascorbic acid at pH=10.4 following the procedure described by Bandulasena et al. (Bandulasena, M.V., Vladisavljevic, G.T., Odunmbaku, O.G., Benyahia, B., 2017. Continuous synthesis of PVP stabilized biocompatible gold nanoparticles with a controlled size using a 3D glass capillary microfluidic device. Chem. Eng. Sci. 171 , 233- 243). The flow rate of ascorbic acid stream was varied from 15 ml/h to 60 ml/h, while the flow rate of FIAuCU solution was kept constant at 15 ml/h. [0098] As shown in Figure 8, the size of AuNPs was precisely controlled in the range from 40 nm to 74 nm by adjusting the flow rate of ascorbic acid stream. A decrease in size of the synthesised AuNPs was achieved by increasing the flow rate of ascorbic acid stream, as can be confirmed by the blue-shift in peak absorbance wavelength, _max (Alzoubi, F.Y., Alzouby, J.Y., Alqadi,

M.K., Alshboul, FI. A., Aljarrah, K.M., 2015. Synthesis and characterization of colloidal gold nanoparticles controlled by the pH and ionic strength. Chinese J. Phys. 53, 100801-100809). A decrease in absorbance at higher flow rates of ascorbic acid solution was due to higher dilution of AuNPs in the product suspension (Bandulasena, M.V., Vladisavljevic, G.T., Odunmbaku, O.G.,

Benyahia, B., 2017. Continuous synthesis of PVP stabilized biocompatible gold nanoparticles with a controlled size using a 3D glass capillary microfluidic device. Chem. Eng. Sci. 171 , 233-243). Smaller NPs achieved at higher flow rates of ascorbic acid stream can be explained by better mixing and lower degree of agglomeration of AgNPs at lower concentration in the dispersion.

Two-phase counter-current flow focusing

[0099] W/O emulsions consisted of droplets of 5 wt% aqueous glycerol solution (p _w = 1012 kg/m ³ and p _w = 1.2 mPa s) dispersed in 2 wt% XIAMETER ^® RSN- 0749 resin in Dow Corning ^® 200 10 cSt fluid (p ₀ = 940 kg/m ³ and m ₀ = 10.4 mPa s) were generated using counter-current flow focusing geometry. The interfacial tension between the aqueous and oil phase was 29.9 mN/m. As shown in Figure 9, the droplet size increased when the flow rate ratio Q _c /Q _d decreased from 4.7 to 2.3, but in both cases highly uniform droplets were achieved. Smaller droplets were formed at higher Q _c /Q _d value due to higher drag force exerted on the liquid interface by the oil phase, which enabled the jet to break up more frequently producing smaller droplets (Ekanem, E.E., Nabavi, S.A., Vladisavljevic, G.T., Gu, S., 2015. Structured biodegradable polymeric microparticles for drug delivery produced using flow focusing glass microfluidic devices. ACS Appl. Mater. Interfaces 7, 23132-23143).

[00100] To investigate the droplet generation stability of the module, an additional formulation of W/O emulsion has been tested for a longer operation time. The oil phase consisting of 0.75% (w/v) 4-aminobenzoic acid dissolved in Miglyol 840 and the aqueous phase consisting of 3% (w/v) Eudragit S100 at pH > 7 have been used to generate W/O emulsion. After droplet formation, 4- aminobenzoic acid diffused to the droplet interface and caused the neutralisation of charged carboxylic groups on Eudragit chains triggering a sol-gel transition of Eudragit S100. Relatively weak 4-aminobenzoic acid (pKa = 2.38) achieved droplet gelation after pinch-off which led to small gel beads. The device was stable and leakproof for more than 6 h and was able to generate highly monodispersed droplets. However, much stronger p- toluenesulfonic acid (pKa = -2.8) caused premature gelation of interface before droplet pinch-off, which resulted in large gel plugs.

Three-phase flow

[00101] Preparation of multiple emulsions: The inner aqueous phase composed of 5 wt% glycerol (p _ip = 1012 kg/m ³ and m _ίr = 1.2 mPa s), the oil phase composed of 2 wt% XIAMETER ^® RSN-0749 resin in 98 wt% Dow Corning ^® 200 fluid { _Pmp = 940 kg/m ³ and p _mp = 10.4 mPa s) and the outer aqueous phase composed of the mixture of 40 wt% glycerol and 2 wt% PVA ( p _op = 1107 kg/m ³ and m _or = 7.9 mPa s) were used to generate W/O/W emulsions by combining co-flow and counter-current flow focusing. The interfacial tension at the inner and outer interface was 29.9 and 31.8 mN/m, respectively. The ability to self-align capillaries was also investigated, as well as the ability to change the distance between inner round capillary orifices while droplets were produced.

[00102] Advantageously, the microfluidic device self-aligns the inner capillaries precisely along the main axis and generate stable flows to make double emulsion droplets with single and multiple cores. The number of internal water droplets in each oil drop was changed from 4 to 2 to 1 by decreasing the inner phase flow rate from 4 to 2.5 to 2 ml/h at constant flow rates of the middle and outer fluids. It was possible to manipulate the shell thickness and the size of internal droplets by controlling the flow rate ratio Q _ip /Q _{mP ·} Furthermore, it was possible to encapsulate simultaneously calcein in the core fluid and Nile red in the shell fluid and to achieve 100 % encapsulation efficiency of both dyes.

[00103] Advantageously, the device of the present invention has the ability to move the injection capillary backward and forward and change the distance AL between the tips of the two inner tubes while the experiment is running. A negative AL value means that the tip of the injection tube is placed upstream of the inlet section of the collection tube. The minimum drop size was achieved when AL/D _C = 0, i.e. when the outlet section of the injection tube coincided with the inlet section of the collection tube. The size of core/shell droplets was manipulated at constant fluid flow rates by changing the distance between the two inner capillaries from -0.8 D _c to zero. It can be explained by the fact that shear force exerted on the outer interface depend on the position of the injection tube. The maximum shear is at the inlet section of the collection tube due to smallest cross-sectional area and the highest velocity of the outer fluid. By pushing the injection nozzle inside the collection tube to achieve AL > 0, it was possible to produce double core droplets and to adjust their size. The greater the AL value, the larger the size of the generated inner and outer drops.

[00104] A three-phase device can be used as a droplet-based microfluidic mixer, which will be demonstrated by the green synthesis of AgNPs within aqueous droplets formed in an inert and environmentally friendly oil. The process is based on merging together 3 mM aqueous solution of silver nitrate delivered through the injection tube and 35 mM aqueous tannic acid solution at pH = 8 delivered co-currently through the outer capillary. The formed mixture was immediately flow focused by Miglyol 840 stream delivered from the opposite side of the outer capillary, which resulted in the generation of mixture droplets in the collection tube. At alkaline pH, tannic acid hydrolyses into glucose and gallic acid and gallic acid induces rapid formation of AgNPs at room temperature (Ahmad, T., 2014. Reviewing the tannic acid mediated synthesis of metal nanoparticles. J. Nanotechnol. 2014, 954206. http://dx.). Smaller

NPs were formed using higher oil flow rates. The size of the synthesized AgNPs was precisely controlled by the size of the reaction droplets. Smaller droplets were formed at higher shear conditions, due to more vigorous mixing within droplets. The advantage of 3-phase device over 2-phase device with two parallel reactant streams is that the reaction mixture is isolated from the walls of the collection tube, which prevents deposition of the NPs onto the walls. [00105] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[00106] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[00107] The invention is not restricted to the details of any foregoing embodiments.

The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. The claims should not be construed to cover merely the foregoing embodiments, but also any embodiments which fall within the scope of the claims.

[00108] Throughout the description and claims of this specification, the words “comprise” and“contain” and variations of them mean“including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[00109] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[00110] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.