The goal of our research is to fabricate an autonomic material system that provides compartmentalization and multi-bilayer networks for enabling collective biomolecular functionality, as is found in living cells and tissues. The material system is based on biomolecular unit cells, which consist of synthetic lipid bilayers formed at the interfaces of lipid-coated aqueous droplets submerged in oil and contained in a solid material. This paper focuses on microfluidic encapsulation of unit cells within a solid material and tuning the amount of contact between droplets, two approaches aimed at increasing the functional density of the droplet-based material system. Hydrodynamic traps within microfluidic platforms have shown to be a promising method to capture single droplets within microfluidic devices. Herein, we develop a resistive flow model to design hydrodynamic traps for collecting pairs of droplets in a direct trapping mode to form unit cells. We also compare to the model the results of droplet trapping in a prototype microfluidic device fabricated prior to model development. In addition to flow techniques for assembling unit cells in solid materials, we examine the use of mineral oil as the hydrophobic oil phase that surrounds the droplets to increase the area of the lipid membrane formed between neighboring droplets. Compared to hexadecane, mineral oil produces larger contact areas between droplets and more-tightly packed multi-bilayer networks. The total free energies of formation for droplet arrays in mineral oil and hexadecane indicate that connected droplets in mineral oil exhibit a greater decrease in free energy upon formation (i.e. they exist at a lower energy state compared to those in hexadecane) and that hexagonal packing provides the maximum amount of decrease in free energy per droplet for droplets in large arrays. Electrical measurements of unit cells formed in mineral oil initially show gigaohm resistances typical of unit cells, however these unit cells exhibit increasing values of conductance as the bilayer areas grow.
This work demonstrates a low-volume microfluidic system that enables rapid assembly of droplet interface bilayers with in situ electrical characterization.
This paper focuses on developing a closed fluidic environment for packaging biomolecular unit cells, which consists of a synthetic lipid bilayer and other biomolecules contained in a near solid-state material with two regions that contain hydrophobic oil (i.e. nonpolar solvent) surrounding aqueous droplets. This research provides a stepping-stone towards an autonomic biomolecular material system, whereby a packaged system will allow for precise droplet interface bilayer (DIB) formation without the interference of outside contamination for long-term applications. Also, substrate materials need to maintain droplets and preserve the self-assembly and stimuli-responsive properties of biomolecules within the unit cell. A critical feature of an encapsulating material is that it does not absorb either of the liquid phases required to form DIBs. Oil depletion tests within sealed, polymeric substrates show that polydimethylsiloxane (PDMS) absorbs full volume of injected hexadecane in approximately 27 hours. However, polyurethane substrates maintain the original amount of oil injected even after several weeks. Bilayer lifetime is also monitored within an environment in which the oil is also depleting. The results of this test show the longevity of a DIB to be shorter than oil lifetime. The lipid-encased droplets disconnect after approximately 10 hours, when there is only approximately <60% amount of oil present. In addition, an initial microfluidic substrate is designed such that a single T-junction intersection can be used to form monodisperse droplets within a primary oil-filled channel and a downstream increase in channel width can be used to connect droplets to form DIBs.
Our research focuses on creating smart materials that utilize synthetic cell membranes assembled at liquid interfaces for autonomic sensing, actuation, and energy conversion. Unlike single membrane assemblies, systems featuring many membranes have the potential to offer multi-functionality, greater transduction sensitivity, and even emergent behaviors in response to environmental stimuli, similar to living tissue, which utilizes networks of highly packed cells to accomplish tasks. Here, we present for the first time a novel microfluidic platform capable of generating a stream of alternating droplet compositions, i.e. A-B-A-B, and sequentially capturing these droplets in precise locations to enable the spontaneous formation of synthetic lipid bilayers between droplets of different compositions (i.e. A and B) in an enclosed substrate. This platform preserves a key feature of the droplet interface bilayer (DIB) method, which allows asymmetric conditions within and across the membrane to be prescribed by simply using droplets containing different species. In this work, we demonstrate the ability to assemble bilayers consisting of asymmetric lipid compositions and, separately, show that alternating droplets containing the same lipid type can also be used to control the direction of ion channel insertion. In the first study, A and B droplet types contain liposomes comprised of different lipid types, which are used to establish an asymmetric composition of the leaflets that make up the lipid bilayer. This asymmetry results in a dc, non-zero membrane potential, which we measure via membrane capacitance versus bias voltage. In the second study, alamethicin peptides are included in only one of the droplet types, which enable voltage-dependent insertion to occur only at one polarity. Cyclic voltammetry measurements are performed to confirm the direction of insertion of alamethicin channels in bilayers. Also, these results show the ability to perform simultaneously electrical measurements on multiple DIB, which increases the experimental capacity and efficiency of a microfluidic approach. The ability to produce alternating droplets in a high throughput manner with electrical access provides a system to investigate the effects of lipid asymmetry on the function of membrane proteins in a controlled model system.
A primary focus of the rapidly growing field of plant synthetic biology is to develop technologies to precisely regulate gene expression and engineer complex genetic circuits into plant chassis. At present, there are few orthogonal tools available for effectively controlling gene expression in plants, with most researchers instead using a limited set of viral elements or truncated native promoters. A powerful repressible-and engineerable-binary system that has been repurposed in a variety of eukaryotic systems is the Q-system from Neurospora crassa. Here, we demonstrate the functionality of the Q-system in plants through transient expression in soybean (Glycine max) protoplasts and agroinfiltration in Nicotiana benthamiana leaves. Further, using functional variants of the QF transcriptional activator, it was possible to modulate the expression of reporter genes and to fully suppress the system through expression of the QS repressor. As a potential application for plant-based biosensors (phytosensors), we demonstrated the ability of the Q-system to amplify the signal from a weak promoter, enabling remote detection of a fluorescent reporter that was previously undetectable. In addition, we demonstrated that it was possible to coordinate the expression of multiple genes through the expression of a single QF activator. Based on the results from this study, the Q-system represents a powerful orthogonal tool for precise control of gene expression in plants, with envisioned applications in metabolic engineering, phytosensors, and biotic and abiotic stress tolerance.
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