On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are ...

Wageningen University & Research

Membraneless organelles formed by liquid-liquid phase separation are dynamic structures that are employed by cells to spatiotemporally regulate their interior. Indeed, complex coacervation-based phase separation is involved in a multitude of biological tasks ranging from photosynthesis to cell division to chromatin organization, and more. Here, we use an on-chip microfluidic method to control and study the formation of membraneless organelles within liposomes, using pH as the main control parameter. We show that a transmembrane proton flux that is created by a stepwise change in the external pH can readily bring about the coacervation of encapsulated components in a controlled manner. We employ this strategy to induce and study electrostatic as well as hydrophobic interactions between the coacervate and the lipid membrane. Electrostatic interactions using charged lipids efficiently recruit coacervates to the membrane and restrict their movement along the inner leaflet. Hydrophobic interactions  cholesterol-tagged RNA molecules provide even stronger interactions, causing coacervates to wet the membrane and affect the local lipid-membrane structure, reminiscent of coacervate-membrane interactions in cells. The presented technique of pH-triggered coacervation within cell-sized liposomes may find applications in synthetic cells and in studying biologically relevant phase separation reactions in a bottom-up manner.

pH-Controlled Coacervate-Membrane Interactions within Liposomes.

Billions of years of Darwinian evolution has led to the emergence of highly sophisticated and diverse life forms on Earth. Inspired by natural evolution, similar principles have been adopted in laboratory evolution for the fast optimization of genes and proteins for specific applications. In this review, we highlight state-of-the-art laboratory evolution strategies for protein engineering, with a special emphasis on in vitro strategies. We further describe how recent progress in microfluidic technology has allowed the generation and manipulation of artificial compartments for high-throughput laboratory evolution experiments. Expectations for the future are high: we foresee a revolution on-a-chip.

(R)evolution-on-a-chip.

Engineering synthetic cells has a broad appeal, from understanding living cells to designing novel biomaterials for therapeutics, biosensing, and hybrid interfaces. A key prerequisite to creating synthetic cells is a three-dimensional container capable of orchestrating biochemical reactions. In this study, we present an easy and effective technique to make cell-sized porous containers, coined actinosomes, using the interactions between biomolecular condensates and the actin cytoskeleton. This approach uses polypeptide/nucleoside triphosphate condensates and localizes actin monomers on their surface. By triggering actin polymerization and using osmotic gradients, the condensates are transformed into containers, with the boundary made up of actin filaments and polylysine polymers. We show that the guanosine triphosphate (GTP)-to-adenosine triphosphate (ATP) ratio is a crucial parameter for forming actinosomes: insufficient ATP prevents condensate dissolution, while excess ATP leads to undesired crumpling. Permeability studies reveal the porous surface of actinosomes, allowing small molecules to pass through while restricting bigger macromolecules within the interior. We show the functionality of actinosomes as bioreactors by carrying out  protein translation within them. Actinosomes are a handy addition to the synthetic cell platform, with appealing properties like ease of production, inherent encapsulation capacity, and a potentially active surface to trigger signaling cascades and form multicellular assemblies, conceivably useful for biotechnological applications.

Actinosomes: Condensate-Templated Containers for Engineering Synthetic Cells.

Biosensing using liquid crystals has a tremendous potential by coupling the high degree of sensitivity of their alignment to their surroundings with clear optical feedback. Many existing set-ups use birefringence of nematic liquid crystals, which severely limits straightforward and frugal implementation into a sensing platform due to the sophisticated optical set-ups required. In this work, we instead utilize chiral nematic liquid crystal microdroplets, which show strongly reflected structural color, as sensing platforms for surface active agents. We systematically quantify the optical response of closely related biological amphiphiles and find unique optical signatures for each species. We detect signatures across a wide range of concentrations (from micromolar to millimolar), with fast response times (from seconds to minutes). The striking optical response is a function of the adsorption of surfactants in a nonhomogeneous manner and the topology of the chiral nematic liquid crystal orientation at the interface requiring a scattering, multidomain structure. We show that the surface interactions, in particular, the surface packing density, to be a function of both headgroup and tail and thus unique to each surfactant species. We show lab-on-a-chip capability of our method by drying droplets in high-density two-dimensional arrays and simply hydrating the chip to detect dissolved analytes. Finally, we show proof-of-principle  biosensing in the healthy as well as inflamed intestinal tracts of live zebrafish larvae, demonstrating CLC droplets show a clear optical response specifically when exposed to the gut environment rich in amphiphiles. Our unique approach shows clear potential in developing on-site detection platforms and detecting biological amphiphiles in living organisms.

Designing Biological Microsensors with Chiral Nematic Liquid Crystal Droplets.

Paper microfluidics has been extensively exploited as a powerful tool for environmental and medical detection applications. Both flow delay and compatibility with either polar or non-polar reagents are indispensable for the automation of detections requiring multiple reaction steps. This article reports the systematic studies of a 3D-printing protocol, characterization, and application of both the partially and fully penetrated polydimethylsiloxane (PDMS) barriers for flexible flow control in paper microfluidics. The physical parameters of PDMS barriers printed using a simple liquid dispenser were found related to the printing pressure, speed, diffusion time after printing, baking temperature, and PDMS viscosity. The capability of PDMS barriers to confine the flow of non-polar solvents was demonstrated using oil flow in both wax- and PDMS-surrounded channels. It was identified that the minimum width of channels to prevent leakage was 470 ± 54 μm, which was as narrow as that fabricated using stamps from lithography. Both the partially penetrated barriers (PPBs) and constriction channels were of the capability to delay flow in paper microfluidics. Additionally, an  investigation led to the further understanding that the reduction of channel cross-section resulting from PPBs was the primary reason for flow delay. Our results suggest that increasing the penetration depth of the barriers is more efficient in delaying flow than increasing the PPB length. Finally, devices with four inlet channels and 0-6 PPBs across each channel were successfully applied in flow delay for sequential fluid delivery. These results improve the understanding of the major factors, affecting the 3D PDMS barrier fabrication and the resulting flow control in paper microfluidics, providing practical implications for applications in various fields.

Siddharth Deshpande

Laboratory of Physical Chemistry and Soft Matter

Wageningen University & Research

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Siddharth Deshpande

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Physical Chemistry and Soft Matter

Wageningen University & Research

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Laboratory of Physical Chemistry and Soft Matter