Figure 1.6 Diagrams representing the basic method of forming droplet interface bilayers (DIBs)
from the contact of two droplets of water in oil in the presence of a bilayer-forming lipid.
Droplet interface bilayers (DIBs) are typically formed from the contact of two aqueous droplets in oil in the presence of lipid, supplied either dissolved in the oil phase or as vesicles in the aqueous solutions (Figure 1.6). Lipid monolayers self-assemble at the oil/water interfaces and bilayers are formed where two monolayers are brought into close proximity92. This method of bilayer formation was first reported many decades
ago, but interestingly only started gaining traction as an ALM study for various applications in the past two decades, notably by Hagan Bayley’s research group at Oxford75 and Takeuchi´s in Tokyo93. Since then, a large variety of applications and
different DIB incarnations have arisen (Figure 1.7). DIBs have been formed between an aqueous droplet and hydrogel surfaces92, between hydrogel shapes94 and between
aqueous droplets in air95. Physical encapsulation of DIBs has also been explored in
31
Figure 1.7 Different methods of forming DIBs (DIBs outlined with dotted line box). In all of the
incarnations, phospholipid is present in the oil phase. a) DIB formed between two aqueous droplets in oil. b) DIB formed between an aqueous droplet contacting a hydrogel surface, submerged in oil. c) DIB formed between two hydrogel shapes in oil. d) Air-stable DIB formed between two aqueous droplets on a surface coated in oil. e) DIBs formed between two aqueous droplets contained within a droplet of oil in water. DIBs also form between the internal droplets and the external aqueous environment.
Notable benefits of DIBs include their stability, reported to be able to survive indefinitely under conditions that prevent evaporation92. They also exhibit a relative ease in
incorporating membrane proteins, with a large variety of prokaryotic and eukaryotic proteins reported to have been introduced and studied in DIBs98-100. The ability to
provide the lipid as vesicles suspended in the aqueous phases allows for the facile production of asymmetric bilayers101, important for the characterisation of biological
membranes that exhibit this property. The use of droplets allows for novel methods to be developed due to their ease of manipulation. For example, the area of DIBs formed from droplets that are anchored on electrodes can be dynamically controlled by moving the electrode102, and reconstituted membrane proteins can be corralled103. DIBs can be
formed and reformed indefinitely between many droplets, allowing for automation techniques104 and multiplexed bilayer formation, which can be employed to give rise to
a)
b)
c)
32
high-throughput assay platforms105. DIBs also lend themselves well to the microfluidic
realm for which numerous droplet manipulation techniques exist104, 106-108 (see section
1.3). For many of these reasons, DIBs have increasingly been used to study membrane proteins99, 100, 109, model cell membranes110, 111 and nanopore sensing112,
with some of these applications involving unprecedented, high-throughput bilayer arrays100, 112.
1.2.4.2.1 DIB Networks
A particular, paradigm-shifting aspect of DIBs is their ability to give rise to networks75, 113, where DIBs are formed between any number of contacting droplets. This provides a
foundation for the use of DIBs as novel devices, and for collective properties to arise and be studied. For example, “biobatteries”75 and wave rectifiers114 have been
produced from DIBs, which offer the potential to give rise to soft matter electronic components. The use of DIBs as logical gate operators has briefly been explored as well115.
The network forming properties of DIBs are of particular interest within the field of bottom-up synthetic biology; in its ability to give rise to biologically inspired multi- compartmentalised structures91. Pioneering work employing this concept was
performed by Gabriel Villar in Bayley´s group, who devised freestanding droplet networks by containing aqueous droplets within a larger oil droplet (Figure 1.8), allowing the formation of DIBs between the aqueous droplets and also between the aqueous droplets and an external, aqueous environment116. These structures, termed
multisomes, have been manufactured via manual methods using micropipettes and by tethering the oil droplet to a silver wire “frame” in water, to avoid the structure from rising and rupturing at the water/air interface. Thus, work is still required in order to generate robust and freestanding multisomes. Work performed by Elani et al. has
33 taken this concept further by producing multisomes using microfluidic methods, and using them as “cell-like reactors”, where ethanolamine in one droplet within the multisome crosses a bilayer into a second, reaction compartment where pyrylium is converted into pyridinium.
Villar et al. also developed droplet printing method which allowed for the creation of DIB network-based “tissue-like materials” (Figure 1.8), formed from thousands of picolitre droplets117. This novel material exhibited mechanical properties comparable to
soft tissues, such as brain tissue, and was able to give rise to selective conductive paths with use of α-Hemolysin pores and self-assembled morphological changes.
34
Figure 1.8 Diagrams depicting different two different DIB technologies developed by Dr. Villar in
Hagan Bayley´s research group. a) the conception of multisome structures, formed from a number of aqueous droplets contained within a larger droplet of oil in the presence of lipid. Bilayers form between the internal aqueous compartments of the multisome and also between the compartments and the external aqueous environment. b) The development of a droplet printing technology allowing for the formation of tissue-like materials consisting of thousands of picolitre droplets forming a large bilayer network. Images are adapted from referenced
publications116, 117.