A new droplet-forming fluidic junction for the generation of highly compartmentalised capsules †

A new oscillatory microfluidic junction is described, which enables the consistent formation of highly uniform and complex double emulsions, and is demonstrated for the encapsulation of four different reagents within the inner droplets (called cores) of the double emulsion droplets. Once the double emulsion droplets had attained a spherical form, the cores assumed specific 3D arrangements, the orchestration of which appeared to depend upon the specific emulsion morphology. Such double emulsion droplets were used as templates to produce highly compartmentalised microcapsules and multisomes. Based on these construct models, we numerically demonstrated a model chemical reaction sequence between and within the liquid cores. This work could provide a platform to perform space/time-dependent applications, such as programmed experiments, synthesis, and delivery systems.


Introduction
Droplet microfluidics 1 , is usually based on liquid droplet formation in another immiscible phase, and can provide large numbers of uniform, independent, and controllable droplets 2,3 .
Such droplets have been widely applied to diverse chemistries 4 , for instance, in d-PCR 5 , organic synthesis 6 , the fabrication of microparticles 7 , and various cell manipulations 8,9 .In addition, droplet microfluidics shows outstanding performance in the generation of consistent multiple emulsions 10 , when compared to conventional batch methodologies, though the volumes produced are often far less.Complex multiple emulsion droplets 11 , can be continuously formed and processed 12,13 , within modular or monolithically integrated, microfluidic devices and systems, that enable the fabrication of micro-scale vesicles/particles with cellular-like, internal structures 14 with certain arrangement 15,16 .Such constructs provide a generic, hierarchical platform, which can enable programmed chemical interactions, that are required for the engineering of (bio)chemistry and synthetic biology [17][18][19][20] .
Droplet forming, fluidic junctions, are important elements in droplet microfluidics, and include the T-shaped junction 21 , the flow-focusing junction 22 (also called the cross-shaped junction), and the co-flow junction 23 .The droplet formation process in these junctions has been well-studied 24 , and indicates that the dynamic flow profiles 25 , the methods to deliver fluids 26 , the channel wettability and their geometries [27][28][29][30] , as well as the liquid phase properties 31 (e.g. chemical composition 32 , dynamic viscosity 33 , interfacial tensions 34 , surfactant composition 35 , suspension components 36 , etc.), can all influence the attained droplet morphology.Generally, the strategy to form multiple emulsions, are to use a series of droplet forming junctions, within either microcapillary-based 37 , or, planar, chip-based, microfluidic devices, by either one-step methods 38 , or multistep methods 39 .The one-step method demonstrates the fine control over the formation of multi-layer droplets 40 , whilst the multi-step method exhibits the ability to generate compartmentalised droplets, by encapsulating multiple inner droplets 41 .(Here, we adopt the terminology, "cores" 42 , to describe the encapsulated droplets, so as to differentiate them from the overall multiple emulsion droplets.)However, due to the existence of hydrodynamic instabilities during droplet breakup, the sequential emulsification process is constrained by the need to synchronise droplet formation at each dropletforming junction 43 .In part, this is because the relatively simple architectures of these classical fluidic junctions, have limited scope to spatially confine fluidic interfacial interactions.Consequentially, by using such simple junction architectures, a feed of a first emulsion, into a new secondary immiscible phase, could result in droplet polydispersity, not only in absolute size of the overall droplet, but also because of the variations of the encapsulated liquid cores.Therefore, generally, to obtain more precision over compartmentalised droplet formation, superior flow rate controls are required, to constrain the control of droplet morphology within a desired narrow range 43 .
In the work presented here, we evaluate a new bifurcated microfluidic geometry (the bat-wing junction), that was designed to form complex multiple emulsions, in a stepwise emulsification mechanism, via its feature of precision flow sectioning.We studied the flow oscillation behaviour at fluidflow bifurcations within the geometry, and studied their influence, on the control of droplet breakup and size.Also, we evaluated this geometry for the generation of double emulsion droplets, where we encapsulated multiple reagents, and evaluated the precision control over the number and relative

Fabrication and setup of the microfluidic device
Circular microfluidic chips (50mm diameter, 3mm thick) were fabricated from polytetrafluoroethylene (PTFE) using a surfacemilling machine (LPKF, C30) fitted with dual-branch, fishtail routers rotating at 30,000 rpm.The resulting rectilinear duct surfaces typically had a roughness (Ra) of ~2um as measured with a VEECO NT3300 white-light interferometer.A 10mm thick ground glass disc coated with a 100 um thick perfluoroalkoxy (Goodfellows, UK) film, was placed over the PTFE disc, so as to provide both a (i) continuous duct geometry with a continuous water contact angle of ~108º and, (ii) a robust viewing window when mechanically compressed within a stainless steel integration and fluidic interface manifold (supplementary figure 1).

Experiment and measurement
In the experiments, water/polymer/oil (W/P/O) was used as a template for the formation of double emulsions and were fabricated using a stepwise emulsification method on a PTFE substrate microfluidic chip, without any PTFE surface treatment.Constant flow-rate, fluid delivery from 10mL gastight syringes (SGE Analytical Science), through PEEK and PFA interconnects, was enabled via syringe displacement pumps (KD Scientific, model 789200L).The morphology of the double emulsion was controlled by the input flow rates, importantly, without adding any surfactant.The core-shell shaped W/P segments were photo-cured, on the fly, within a length of PTFE outlet tubing (1.5mm inner diameter) by exposure to a UV light source (5W 365nm-wavelength UV LED, placed 1cm away from the tubing).Solid samples were collected in a beaker filled with stirred carrier phase, and washed three times with acetone before analysis.Real-time video was recorded to study the droplet formation using a high-speed (400fps) camera (MS40KD2C1, Mega Speed Corporation) mounted on a Nikon AZ100 microscope.Dimensional images of solid capsules were recorded using a Nikon MM-800 measuring microscope, and analysed with Nikon NIS-elements D 3.2 software.

Computational fluid dynamics simulation
Three-dimensional, time-dependent, computational fluid dynamics simulations, were developed to model the droplet breakup process at the bat-wing junction, by using COMSOL Multiphysics 5.0 software (COMSOL Inc., Burlington, MA).The isosurface, volume arrow, velocity magnitude and pressure distributions were plotted as geometric median surfaces to demonstrate the droplet breakup process.Numerical data were obtained for the analysis.

Description of bat-wing junction geometry and operation
The structures of droplet forming junctions, play an important role in droplet formation, due to the influence of spatial constraints on the delivery, and the interactions, of the fluids employed.The bat-wing junction was designed to comprise two cross-shape intersections, aligned end-to-end, and sharing the side inlets with bifurcations as shown in Figure 1a.The two intersections were linked by an expansion zone, and terminate with an expansion outlet.These width-expanding structures were utilised to focus and dissipate the fluid flows, and create local maximum velocity magnitude points, where the highest shear force occurs.The upstream and downstream bifurcations couple the pressures at the two intersections, and form a uniform pressure gradient, within the expansion zone.The widths of the bifurcations are narrower than the central channel, so as to be easily obstructed by the middle flow.The staggered geometry serves as a fluid shunt and directs the fluid delivery, either upwards or downwards, according to the local dynamic pressure distribution.

Single emulsion formation
Similar to other droplet forming junctions, three droplet formation regimes were observed when using the bat-wing junction.As shown in Figure 1b, the two left images show the water droplet (blue) break-up in mineral oil (red), in both dripping and squeezing regimes.The three images to the right, demonstrate the TMPTA droplet (red) break-up, in mineral oil (transparent), both in jetting, dripping and squeezing regimes, respectively.Three-dimensional, computational fluid dynamics simulations, were conducted to study the details of the droplet formation at the bat-wing junction (Figure 1c).It was found that the continuous phase flow was predicted to oscillate between the upstream and downstream bifurcations.This oscillation was related to the dispersed phase position, inside the expansion zone, during droplet formation (Figure 2a&b, supplementary video 1).Continuing with the droplet formation process, while the dispersed phase (only) blocks the upstream bifurcation, the local pressure at the blockage, starts to increase, forming a vortex, and most of continuous phase flows through the downstream bifurcation.If the incoming dispersed phase blocks both the upstream and downstream bifurcations, the continuous phase is squeezed within the expansion zone that lies between the upstream and downstream bifurcations.The continuous phase accumulates in the expansion zone, squeezing the dispersed phase into an hourglass form, which is eventually caused to break off, or section.Since the volumes of Please do not adjust margins Please do not adjust margins continuous phase from the upstream and downstream bifurcations flowing into the expansion zone, vary dramatically during a droplet formation cycle (Figure 2c), this in turn induces different flow patterns during droplet breakup (Figure 2d).Hence, the droplet breakup point, as well as the droplet size, could be controlled by the continuous phase inflow rate (Figure 2e).This feature of the bat-wing junction was utilised as a precise packaging tool, to assemble uniform, dispersed phase segments of definable length, during the emulsification process within the microchannel.It was also found that a passive satellite droplet removal mechanism resulted from the squeezing regime, within the batwing junction.This was due to the incoming dispersed phase blocking the upstream bifurcation, just after the droplet breakup, resulting in the continuous phase being forced through the downstream bifurcation.Any formed satellite droplets were found to remain at a stationary position inside the expansion zone, and effectively merged with the incoming dispersed phase, due to the surfactant-free emulsion templates (supplementary video 2).

Double emulsion droplet formations
The double emulsion droplet formation is more complicated than for the single emulsion, since the encapsulated water droplets within the first emulsion offer more resistance during breakup, due to the higher interfacial tension of water, over that of water and dispersed phases.For the stepwise emulsification method, the existence of multiple hydrodynamic instabilities will influence the monodispersity of the final multiple emulsion droplets.In the next two sections, we demonstrate the performance of the bat-wing junction in forming uniform double emulsion droplets using a two-step emulsification method.
Monodispersed core-shell shaped droplets.Here, we investigated the formation of double emulsions, using the bat-wing junction as a second droplet-forming junction, to section the first emulsion, in a two-step method.As shown in Figure 3a, the first emulsion was formed at the first cross-shaped, flow-focusing junction, as the inner water phase (blue, from middle) droplets, were broken-up in the TMPTA flow (transparent, from sides).The shell phase inflow rate was maintained at a constant 0.2ml/hr., and the inner water phase inflow rate was increased from 0.05ml/hr., 0.1ml/hr.… to 1.4ml/hr., in 0.1ml/hr.steps, from left to right (Figure 3a), respectively.These combinations formed different lengths of the first emulsion segment, and it was found that by changing the continuous phase inflow rates (left transparent, right red) at the bat-wing junction, monodispersed, core-shell shaped, double emulsion droplets, could be precisely sheared off, or sectioned, in various volume ratios, between the core and shell phase.Next, we tested the consistency by which, the bat-wing junction formed core-shell shaped, double emulsion droplets, by measuring both their yield rates, and size distributions.As shown in Figure 3b, while the inflow rates ratio, of inner/shell phases were held constant, the yield rates of double emulsion droplets at the outlet, were elevated by increasing the total inflow rate.The variations in time intervals between adjacent droplets entering the outlet, were ±4.84%, ±4.74% and ±3.47%, for the three groups in Figure 3b, from left to right, respectively.At the same time, the sizes of the double emulsion droplets were reduced, while the total inflow rate was increased.The size variations (n=100 for each group) of the inner core droplets was ±2.47%, ±2.01%, ±1.58%, and for the total double emulsion droplets, was ±1.85%, ±1.85%, ±1.86%, from low to high inflow rates.These results suggested that the bat-wing junction could form highly replicated, core-shell shaped, double emulsion droplets, by a two-step method under different inflow rate combinations.Compartmentalized double emulsion droplets.Furthermore, the bat-wing junction could be used to precisely control the number of the encapsulated droplet cores within the double emulsion droplets.This was achieved by forming a consistent first emulsion, with repeated droplet patterns, and then, precisely sectioning them, at the bat-wing junction.As shown in the rest of Figure 3, the inflow rates of the inner and shell phases, were kept constant, resulting in the formation of two strings of droplets in the shell phase flow, as the first emulsion.By changing only the continuous phase flow rates, the number of droplets (which became the inner cores of the double emulsion) could be precisely tuned to a resolution of one core, as shown in Figure 3c.This resolution could be maintained for up to 15 inner cores, as shown in the supplementary video 3.In addition, once the shape of the double emulsion droplets had changed from segments (Figure 3d) to spheres (Figure 3e), the inner droplet cores self-arranged into specific 3D geometric orchestrations, which appeared to depend upon the number of inner cores that were incorporated within the double emulsion.Multiple types of encapsulated droplets.Several groups of control experiments were conducted to form complex double emulsions, that encapsulated multiple types of inner droplet cores, within a single, shell-phase matrix.These were achieved by using two T-junctions, to generate two independent, repeating droplet groups (to become inner cores), within the shell phase flow, as the first emulsion.The patterns of these droplet groups (reagents, sizes and orders), were controlled by the inflow rate combinations.Then, the first emulsions were precisely sectioned off, to form patterned double emulsions.This sectioning encapsulated the first emulsion groups of inner droplet cores, the number of which, could be determined by simply tuning the continuous phase flow rates, to change the droplet breakup points within the bat-wing junction.The groups of inner droplet cores could include either even (Figure 4a-c) or odd (Figure 4d), numbers of such cores.As the double emulsion segments attained a spherical form, the inner droplet cores also arranged into certain geometric orchestrations, as shown in Figure 4e.It appears that such core arrangements within the double emulsion droplets, may possibly be determined by either, or both, their contiguous orders, and their sizes, within the first emulsions.In addition, a droplet forming array, which included four T-junctions as shown in supplementary figure 2, was used to produce more complicated, repeated patterns of the inner droplet core assemblies, in the shell phase flow, as the Please do not adjust margins Please do not adjust margins first emulsion.As shown in Figure 4f & 4g, the bat-wing junction was used to consistently section off double emulsion droplets, each encapsulating four different types of inner droplet cores.The sizes and numbers, of the inner droplet cores, were controlled by the inflow rate combinations.As shown in Figure 4j, by changing the inflow rates of the inner phases, which were 0.2/0.2/0.7/0.7 (ml/hr., inlet combination 1), 0.4/0.4/0.5/0.5 (ml/hr., inlet combination 2), 0.6/0.6/0.3/0.3 (ml/hr., inlet combination 3), the numbers of each coloured inner droplet cores, were controlled, whilst the total number of cores remained in the range of 15-20.This was due to that the (i) shell phase and continuous phase input flow rates, were kept at a combined total of 0.8/2.0(ml/hr.)for all the three combinations, (ii) total first emulsion flow to the bat-wing junction remained at a constant flow rate, and (iii) continuous phase, precisely sectioned the first emulsion to achieve repeated patterns.The variations in the core number partially resulted from the sizes variation of the different coloured water droplets, which could be reduced by higher precision pumping mechanisms.As these double emulsion droplets attained spherical forms, the inner droplet cores also self-arranged into specific 3D geometrical orchestrations, within the shell phase matrix (Figure 4h & 4i).

Microcapsules and multisomes
So far, we have demonstrated how to form highly uniform double emulsion droplets that encapsulate multiple types of inner droplet cores, with controllable morphology, using the bat-wing junction and using a stepwise, emulsification method.The 3D geometrical arrangement of inner droplet cores appeared to depend both on their number, size, and the specific contiguous order, within the first emulsion.These double emulsion droplets could represent very interesting templates, to fabricate, highly compartmentalised, micro-constructs with given functionalities.Figure 5a-r shows solid polymeric microcapsules, which were solidified from the double emulsion droplets, and produced, using the methodologies described above.Once the droplets had attained an approximate spherical form, the shell phase was photopolymerised, on-thefly, using a 365nm UV light source.The photopolymerised shells formed an effectively, fixed encapsulating matrix, within which the inner droplet cores, containing chemical reagents, were held in a constant, 3-dimensional geometrical orchestration.This fixation, thereby, enabled subsequent metrological analysis.Such multicore microcapsules, with a liquid encapsulation matrix, may be used to enable the multiplereagent release, or enable in-situ chemical synthesis in relation to external stimuli.Such encapsulation matrices could include a wide range of degradable polymers (e.g.Polylactic acid), semipermeable materials (e.g.NOA61, as used here), or, employ bilayer lipid membranes decorated with, functional inter-core, or inter-droplet proteins.In contrast to the aforementioned constructs fixed within a photopolymerised polymer, 'soft' analogues (e.g.multisomes) were formed from water/squalene/TMPTA emulsions (Figure 5s-v) or water/squalene/3% alginate emulsions (Figure 5w-y), using the same methods as used before, but on PMMA-based microfluidic chips.The numbers of the inner water droplets were controlled, and the lipids, which were pre-suspended in squalene, were assembled around them to form monolayers. Once the double emulsion droplets had attained spherical forms, the inner droplet cores, again self-arranged, and high-order, 3D droplet interface bilayer networks were established.Such artificial vesicles hold great potential for biotechnology developments and medical applications 44 .
Finally, we simulated model, sequential reactions, and molecular diffusion within a typical multicore construct.This model consisted of 3D inner droplet arrangements, that were based on the practical experiments demonstrated in this paper.Figure 6a modelled a gas capture process within a 13-core, gaspermeable microcapsule, that encapsulated the active reagents.The chemical reaction within the central core was the final reactant in this chain reaction, due to the specific geometrical arrangement of cores.Figure 6b models the sequential chemical reactions within a multicore multisome, that contained four types of cores, each with a different reagent.The diffusion of the water-soluble reagents, was influenced by the bilayer positions, which was also depended upon the inner droplet core arrangements.The reactions took place while the molecules diffused within the multisome.These demonstrations may provide the beginnings of more precise engineering tools for programmed chemistry and synthetic biology.

Conclusions
In summary, we demonstrate a novel, droplet-forming fluidic junction, for which we coined the term, the 'bat-wing junction'.Its bifurcation structures, oscillate the side flows during droplet formation, and the droplet break-up point and droplet size were regulated, by adjusting only the continuous phase inflow rate.We also found that the junction acted as a passive satellite droplet removal mechanism, due to its inherent flow confinement behaviour.This junction can be used in stepwise emulsification methods, to precisely section a patterned, first emulsion, and then form uniform double emulsion droplets, which can encapsulate inner cores (droplets), of various sizes and reagent composition.It was found that once the double emulsions attained a spherical form in the capillary, the inner core self-arranged in certain 3D geometric orchestrations, which appeared to depend upon their sizes, numbers and orders, within the double emulsion droplets.We produced highly compartmentalised microcapsules and multisomes from these double emulsion droplets, on the fly, to inherit the formed droplet morphologies.We further demonstrated by numerical simulation, how these microconstructs could possibly enable sequential chemical reactions, as a generic platform for diverse applications, ranging from (bio)chemical synthesis 45 /analysis 46 , cell passage 47 , smart drug delivery 48,49 , computation 50 and self-repair materials 51 , and artificial cells 52 .
Please do not adjust margins Please do not adjust margins Certain limitations exist in the work reported here that could be explored further, including critically, the spatial constraints due to the 2.5D nature of the planar bat-wing junction used in this study.For this, a truly 3-dimensional, bat-wing junction could be manufactured by the adoption of novel additive manufacturing methods, such as 3D-printing 53,54 .This could enable the more exact sectioning of a first emulsion into even more accurately defined, internally compartmentalised capsules.Also, additional precision control of individual cores (size, order, reagent concentration, etc.) within the first emulsion (before sectioning as double emulsion droplets), could be found by employing pulseless, fluid propulsion mechanisms.Further, sequential droplet processing, could be enabled by liquid carrier phase exchange 55 , vesicle/particle surface overcoating 56 , and non-invasive in-flow droplet characterizations 57 .Such improved characterisations, could be enabled through non-invasive on-the-fly, interferometric or tomographic methodologies, and could enable the increased resolution of individual core droplet volumes and dimensions.
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