We report on microfluidic strategies to generate several multi-compartment membrane-based structures, including droplet interface bilayer networks and multi-compartment vesicles. These developments allow the current status quo— where microdroplets are used as isolated vessels— to be changed. By linking droplets together with lipid membranes, higher order systems can be generated, with particular ramifications for bottom-up synthetic biology and for functional droplet-based microreactors and biodevices

Ces, O

Elani, Y

Law, R

English

Spiral - Imperial College Digital Repository

MICROFLUIDIC GENERATION OF NETWORKED DROPLET COLLECTIONS AND LIPID MEMBRANE CONSTRUCTS    Yuval Elani1, Robert V. Law1 and Oscar Ces1* 1Institute of Chemical Biology, Imperial College London, UK  ABSTRACT We report on microfluidic strategies to generate several multi-compartment membrane-based structures, including droplet interface bilayer networks and multi-compartment vesicles. These developments allow the current status quo— where microdroplets are used as isolated vessels— to be changed. By linking droplets together with lipid membranes, higher order systems can be generated, with particular ramifications for bottom-up synthetic biology and for functional droplet-based microreactors and biodevices.    KEYWORDS: Lipid Vesicles, Droplet Interface Bilayers, Microreactors, Artificial Cells, Synthetic Biology   INTRODUCTION The growth of microdroplet technologies in recent years has led to applications ranging from high throughput screening of genetic libraries, to them being used as chemical reactors for nanoparticle and small-molecule synthesis.[1, 2]  To date, however, most research has involved isolated single-droplet systems. As the field matures there is an increasing need to move towards integrated multi-droplet collections: this could see the introduction of more complex, higher-order, and ‘emergent’ functional properties, to expand the power and scope of microdroplet technologies.  This need is particularly relevant in two disciplines where microdroplets are showing increasing promise. The first is in bottom-synthetic biology: droplets can be considered artificial cells due to their cell-like size, and their capacity to be functionalised with biological components.[3] If single droplets can be used as cellular models, then droplet-collections can likewise be considered multi-cellular entities (e.g. tissues). The second is for the field of soft bio-inspired devices: droplet collections can be designed to function as modular, responsive multifunctional devices, each droplet designated to a particular function. These have potential use for therapeutic, bio-sensing, and microreactor applications.  We report on our efforts to form interlinked droplet networks. We use inspiration from nature to delineate compartments, by employing lipid membranes. This allows the incorporation of biological machinery, in particular transmembrane and membrane-associated proteins to enable compartment communication, and allow for the incorporation of complex life-like functionalities. We develop two such systems: droplet interface bilayer (DIB) networks,[4] and multi-compartment vesicles.[5] These differ from one another by the nature of the external phase (water or oil), and by their method of generation (Figure 1).   Figure 1:  (A) Droplet interface bilayer networks existing in bulk oil (B) Two--compartment vesicles with inner bilayer delineating the compartments, and external bilayer separating the aqueous compartments from the exterior.  978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001 677 18th International Conference on MiniaturizedSystems for Chemistry and Life SciencesOctober 26-30, 2014, San Antonio, Texas, USAEXPERIMENTAL AND RESULTS The microfluidic techniques used to generate the different types of membrane systems all use water-in-oil (w/o) droplets as a starting point, with lipid dissolved in oil. This results in monolayer self-assembly: polar headgroups face the aqueous phase; fatty-acid tails face the oil phase. Where lipid mono-layers meet—including at all droplet/droplet interfaces—a lipid bilayer (i.e. membrane) is formed. A summary of the generation strategies and results are shown in Figure 2.   Figure 1: (A) 3D and 2D bilayer networks were formed by feeding droplets into microfluidic channels. 3D and 2D network architecture was determined by droplet packing geometries and the contours of the microfluidic channels respectively.  (B) (i) Multi-compartment vesicles were generated by phase transfer of a set number of w/o droplets. (ii) Compartment number was defined by ejecting a set number of drop-lets. Here, 2/3/4 compartments are shown. (iii) Compartment contend was defined by the initial droplet content. Different geometrical assemblies were constructed. Here, both planar and tetrahedral 4-compartment vesicles are shown.  To generate DIB networks, microdroplets were generated off-chip and fed into a PDMS microfluidic de-vice. To produce 3D bilayer networks, droplets were packed into a wide 800 µm channel. Depending on the droplet size, distinct packing geometries—and hence network architectures—were generated.  De-fined 2D architectures were formed by confining the droplets in channels of set geometries (in the exam-ple above, ‘Y’ shaped channel). Precise locations of individual droplets were controlled using negative pressures from the branched channel outlets. To generate multi-compartment vesicles, a set number of pre-formed lipid-coated water-in-oil droplets were driven though a water/oil column with an interfacial monolayer. Droplets were loaded with sucrose, and as such were heavier that both phases. Droplets sunk through the interface together thus be-ing encased by a bilayer. Multi-compartment vesicles were generated, where compartment number was determined by how many droplets were driven through the interface together. It is important to note that this was done will full user-control. For example, by ejecting three droplets at once, three compartment vesicles were formed. In addition, by using droplets of different composition, individual compartment content could be specified. Finally, defined geometrical architectures could be assembled.    678DISCUSSION  These systems are constructed using microfluidic technologies, ensuring high throughput generation and narrow size distribution of compartments. Crucially, in both cases, network architecture, connectivity, and droplet composition and size were controlled. Both platforms have distinctive features associated with them. DIB networks are present in bulk-oil: as such they do not exist in physiological setting, so applications for in vivo use are limited. However, as they consist of several interconnected compartments separated by membranes they can be used as multi-cellular mimics, as others have demonstrated.[6]  Multi-compartment vesicles on the other hand exist in a bulk aqueous environment. They therefore have greater potential as droplet-based devices for physiological use. They also useful in a synthetic biology setting, by allowing organelle function to be mimicked: different compartments can be dedicated to perform for specific tasks. We are currently working on introducing signalling cascades for intra-vesicle communication and environmental sensing, thus demonstrating their biomimetic functionality.    CONCLUSION We report microfluidic generation strategies of two types of membrane-based compartmentalised structures: DIB networks and multi-compartment vesicles. In both cases, network architecture, size, and compartment content could be precisely defined. By utilising the power of droplet networks these structures have potential use for applications in bottom-up synthetic biology and for droplet-based microreactors or devices.   ACKNOWLEDGEMENTS This work was supported by EPSRC via grants EP/J0175666/1 and EP/G00465X/1and by an EPSRC Centre for Doctoral Training Studentship from the Institute of Chemical Biology (Imperial College Lon-don) awarded to YE.   REFERENCES  [1] Huebner, A., Sharma, S., Srisa-Art, M., Hollfelder, F., & Edel, J. B. (2008). Microdroplets: a sea of applications?. Lab on a Chip, 8(8), 1244-1254. [2] Theberge, A. B., Courtois, F., Schaerli, Y., Fischlechner, M., Abell, C., Hollfelder, F., & Huck, W. T. (2010). Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and bi-ology. Angewandte Chemie International Edition, 49(34), 5846-5868. [3] Takinoue, M., & Takeuchi, S. (2011). Droplet microfluidics for the study of artificial cells. Analyti-cal and bioanalytical chemistry, 400(6), 1705-1716. [4] Elani, Y., A.J. deMello, Niu, X., & Ces, O. (2012). Novel technologies for the formation of 2-D and 3-D droplet interface bilayer networks. Lab on a Chip, 12(18), 3514-3520. [5] Elani, Y., Gee, A., Law, R. V., & Ces, O. (2013). Engineering multi-compartment vesicle networks. Chem. Sci., 4(8), 3332-3338. [6] Villar, G., Graham, A. D., & Bayley, H. (2013). A tissue-like printed material. Science, 340(6128), 48-52.  CONTACT  * O. Ces; o.ces@imperial.ac.uk 679

Microfluidic generation of networked droplet collections and lipid membrane constructs

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Microfluidic generation of networked droplet collections and lipid membrane constructs

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