General discussion

2.4.1 Fabrication of a novel, hybrid 3D-printed glass capillary device for

the generation of hierarchically assembled higher order emulsions

Using devices fabricated with surface modified glass capillaries, a 3D-printed assembly, and fluidic tubing and connectors, it was possible to produce W/O/W and W/O/W/O emulsions in a facile and cost-effective manner. Glass capillaries displayed

100 ms intervals

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Emulsions.

an effective hydrophilic surface modification allowing for the prolonged generation of oil droplets in water; the 3D-printed scaffold aided in the coaxial assembly of the different hydrophilic and hydrophobic channels; and fluidic connectors such as fingertight fittings demonstrated good compatibility with the 3D-printed scaffold in the ability to interface without any leaks.

Devices were constructed for the formation of W/O/W emulsions via the generation of droplets of W/O and its subsequent coaxial flow with an external aqueous fluid, which pinched-off W/O emulsions into W/O/W emulsions. A second, similar device constituting two coaxial droplet-generating geometries allowed for the subsequent pinching off of W/O/W emulsions in oil, forming W/O/W/O emulsions. This method of hierarchical droplet encapsulation offers the potential to add even more emulsification stages, aided via a basic 3D-printed scaffold which can be added and aligned with each other in series.

A particular benefit of the microfluidic devices designed here are their accessibility, ease of use and cost. The devices are assembled from common laboratory materials. For example, glass capillaries are cheap and are used for electrophysiology applications; and fingertight fittings, ETFE junctions and FEP tubing are commonly found as part of high performance liquid chromatography (HPLC) rigs. 3D-printers are likely to be accessible to many research institutions and can be remarkably low cost. 3D-printing also offers the ability for designs to be digitally shared which aids in the accessibility of the devices to other researchers. Specialised equipment or clean room environments are not required except for the surface modification techniques, which require the use of a plasma cleaner. However, as described in section 2.2.1.1, alternatives surface modification techniques exist. All of this serves to increase the ease via which researchers can produce double or triple emulsions for diverse applications, overcoming the microfluidic fabrication barrier28_.

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2.4.2 Stability

The stability of W/O/W and W/O/W/O was not thoroughly assessed as the experiments focused on the ability to generate such emulsions. However, W/O/W emulsions were observed to remain intact for at least 1 hour when contained in a glass channel, likely due to the presence of surfactants. This was not true for the case of W/O/W/O emulsions, where the internal oil phase would coalesce with the external oil phase. This is likely due to the compound effect of the emulsions being relatively large and the density difference between the inner oil and outer water phase, causing the inner oil phase to rise in relation to the outer water phase. It is likely that this can be circumvented via the solidification of the outer shell of the emulsion. It remains unknown whether emulsions stabilised with lipid instead of surfactants will be sufficiently stable for droplets to survive the mechanical stress provided by droplet- generating junctions. This is explored in the following chapter. However, there is evidence that that lipid bilayers can form in the presence of Span-80 surfactant, which has been reported to become excluded from a DIB once it has formed73_{. This can}

provide an alternative route to producing stable encapsulated DIBs if lipid alone fails to stabilise the emulsion interfaces, although further assessment of the mechanism and effectiveness of surfactant exclusion from lipid bilayers would be required.

2.4.3 Monodispersity

The monodispersity of the aqueous droplets, W/O/W and W/O/W/O emulsions remains untested for these experiments. It was observed that droplets were relatively monodisperse, and for droplet volume and frequency of generation as explored in section 2.3.2.1, low standard deviations were obtained (n = 5 for all experiments and conditions). Due to the geometrically-controlled regime of droplet formation and reports in the literature it is likely that high monodispersity is achievable.

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Emulsions.

2.4.4 Emulsions Size

The emulsions produced here are in the millimetre range and therefore larger than desirable for many most applications including the generation of encapsulated droplet interface bilayers. For this particular application, smaller hierarchical emulsions would allow for higher surface area to volume ratios as well as bilayers of a smaller surface are which would likely increase the stability of the constructs, cheaper experiments due to less usage of reagents, increased efficiency of chemical reactions that require the maintenance of chemical concentrations, as well as the potential to use these constructs as parenteral medical devices for drug delivery, for example. Similar fluidic set-ups for the generation of hierarchical emulsions are seen in the literature, which operate in the micrometre range, giving rise to double emulsions that are 100 - 500 µm in diameter12, 74_.

The geometrically-driven nature of droplet formation, alongside the ability to purchase glass capillaries in the micrometre range and easily scale-down the size of the 3D- printed assembly, mean that it is very likely that the devices presented in this chapter could be scaled down in order to produce smaller emulsions in the ≈100 μm range. This would be the focus of future experiments.