A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models.

Funder: University of CambridgeFunder: Blavatnik Family FoundationCell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing solution. Using our purification approach, we show that giant vesicles can be separated from various residues─which range in size and chemical composition─with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time

Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones.

Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug - we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.The work was supported by an ERC Consolidator grant “DesignerPores” awarded to UFK. JC acknowledges support from the BBSRC. MS was supported by the Friedrich Naumann Foundation for Freedom and the Swiss- European Mobility Programme. KAN was supported by the Erasmus Plus student exchange programme. SHA is supported by a Herchel Smith Postdoctoral Fellowship. SP acknowledges support from the Leverhulme Trust through an Early Career Fellowship (ECF-2013-444).This is the final version of the article. It first appeared from Nature Publishing Group at http://dx.doi.org/10.1038/srep32824

Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner.

Bacterial cells are critically dependent upon pH regulation. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of Escherichia coli. Indole is an aromatic molecule with diverse signalling roles. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry. We show that under conditions permitting indole production, cells maintain their cytoplasmic pH at 7.2. In contrast, under conditions where no indole is produced, the cytoplasmic pH is near 7.8. We demonstrate that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, we illustrate that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a "memory" to maintain the cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.The Leverhulme Trus

Aerosol-jet printing facilitates the rapid prototyping of microfluidic devices with versatile geometries and precise channel functionalization.

Microfluidics has emerged as a powerful analytical tool for biology and biomedical research, with uses ranging from single-cell phenotyping to drug discovery and medical diagnostics, and only small sample volumes required for testing. The ability to rapidly prototype new designs is hugely beneficial in a research environment, but the high cost, slow turnaround, and wasteful nature of commonly used fabrication techniques, particularly for complex multi-layer geometries, severely impede the development process. In addition, microfluidic channels in most devices currently play a passive role and are typically used to direct flows. The ability to "functionalize" the channels with different materials in precise spatial locations would be a major advantage for a range of applications. This would involve incorporating functional materials directly within the channels that can partake in, guide or facilitate reactions in precisely controlled microenvironments. Here we demonstrate the use of Aerosol Jet Printing (AJP) to rapidly produce bespoke molds for microfluidic devices with a range of different geometries and precise "in-channel" functionalization. We show that such an advanced microscale additive manufacturing method can be used to rapidly design cost-efficient and customized microfluidic devices, with the ability to add functional coatings at specific locations within the microfluidic channels. We demonstrate the functionalization capabilities of our technique by specifically coating a section of a microfluidic channel with polyvinyl alcohol to render it hydrophilic. This versatile microfluidic device prototyping technique will be a powerful aid for biological and bio-medical research in both academic and industrial contexts

A synthetic enzyme built from DNA flips 107 lipids per second in biological membranes.

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membrane's inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics.

Funder: Winton Programme for the Physics of SustainabilityFunder: Cambridge-NPL studentshipFunder: Trinity-Henry Barlow ScholarshipFunder: Department for Business, Energy and Industrial Strategy, UK Government; doi: http://dx.doi.org/10.13039/100011693Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions-their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide's physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials

Switching cytolytic nanopores into antimicrobial fractal ruptures by a single side chain mutation

Disruption of cell membranes is a fundamental host defense response found in virtually all forms of life. The molecular mechanisms vary but generally lead to energetically favored circular nanopores. Here, we report an elaborate fractal rupture pattern induced by a single side-chain mutation in ultrashort (8–11-mers) helical peptides, which otherwise form transmembrane pores. In contrast to known mechanisms, this mode of membrane disruption is restricted to the upper leaflet of the bilayer where it exhibits propagating fronts of peptide-lipid interfaces that are strikingly similar to viscous instabilities in fluid flow. The two distinct disruption modes, pores and fractal patterns, are both strongly antimicrobial, but only the fractal rupture is nonhemolytic. The results offer wide implications for elucidating differential membrane targeting phenomena defined at the nanoscale

A Lab-on-a-chip System for the Standardised Characterisation of Membrane Active Antimicrobials

The continuing evolution of bacterial resistance against commercially available antibiotics is recognised as a global health threat, and the discovery of novel antimicrobials is urgently required. Antimicrobial peptides (AMPs) are emerging as important players in the fight against antibiotic resistance. In parallel, the field of microfluidics is maturing, and its benefits are being exploited in applications related to biomimicry and standardised testing. Developing a fundamental understanding of the modes of action of AMPs against membrane models is critical for developing these compounds into novel therapeutics. Throughout my studies, my efforts have been focused on developing microfluidic platforms that can streamline the evaluation of membrane active peptides in a systematic and standardised manner. As a result, I present the "GUV Studio", a bespoke multilayer microfluidic platform to quantify membranolytic efficacy and characterise the mode of action of AMPs. The platform is a biomimetic vesicle-based screening assay, which integrates an element for the high-throughput generation of Giant Unilamellar Vesicles (GUVs) in physiological salt concentrations on demand. Thousands of GUVs are individually immobilised downstream in hydrodynamic traps connected to separate perfusion inlets that facilitate the total fluid exchange of the solutions surrounding the vesicles, and enable the controlled, continuous administration of peptides for 8 different experiments in parallel. Membranolytic activity is expressed as a function of the time needed for an encapsulated dye to leak out of individual GUVs as a result of membrane permeabilisation or lysis. The platform has been used to study 20 native and de novo synthesised peptides at different concentrations. The results generated provide novel insights into the activity of a range of membrane active peptides, and demonstrate the capability of the lab-on-a-chip system to differentiate various modes of action defined by the response of the vesicle population to the peptides. My platform provides a quantitative, high-resolution tool to investigate the activity of any membrane-active compounds in a controlled, highly parallelised and high-throughput manner.Cambridge-National Physical Laboratory (U.K.) studentship, the Winton Programme for the Physics of Sustainability, the Trinity-Henry Barlow Scholarship, and the ERC (Designer-Pores 647144

Research data supporting "Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner"

This dataset was collected by Fluorescence Spectroscopy and FlowCytometryJinbo Zhu thank the support from UK Engineering and Physical Sciences Research Council (EPSRC, EP/M008258/1). Kareem Al Nahas acknowledges support from the Winton Programme for the Physics of Sustainability, Trinity-Henry Barlow Scholarship, National Physical Laboratory and ERC consolidator grant (DesignerPores 647144)

Measuring Thousands of Single-Vesicle Leakage Events Reveals the Mode of Action of Antimicrobial Peptides.

Funder: Phospholipid Research CenterFunder: Engineering and Physical Sciences Research CouncilFunder: Trinity College, University of CambridgeFunder: Department for Business, Energy and Industrial Strategy, UK GovernmentHost defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, classical characterization assays often lack the ability to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations and related the observed modes to the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner and for assigning specific modes of action to the corresponding antimicrobial mechanisms