Microfluidic platform for bilayer experimatation from a research tooltowards drug screening

The aim of this thesis, which is the development of a microfluidic platform for bilayer experimentation with the potential for drug screening on ion channels, is introduced in this chapter. After a short presentation of the field of drug screening, an outline of this thesis is given, together with a brief summary of the different chapters

Proliposome formulations for delivery via medical nebulisers

This study aims to investigate the ability of proliposomes to generate liposomes for delivery using air-jet, ultrasonic and vibrating-mesh nebulisers. Particulate-based proliposomes successfully generated liposomes under static conditions. Manually dispersed proliposomes generated multilamellar vesicles, with formulation having a small effect on the liposome size. Using sucrose as a carrier, liposomes were generated or dispersed in situ from proliposomes within the medical nebulisers investigated. The Pari (air-jet) and the Omron (vibrating-mesh) nebulisers produced large mass and lipid outputs with a large lipid fraction deposited in the lower stage of a two stage impinger. The Liberty (Ultrasonic) nebuliser failed to deliver more than 6% of the lipid employed. Multilamellar liposomes were generated from ethanol-based proliposomes. The resultant vesicles entrapped 62% of the available salbutamol sulphate compared to only 1.23% entrapped by liposomes made by the thin film method. Aeroneb Pro or Aeroneb Go vibrating-mesh nebulisers generated aerosol droplets of larger volume median diameter and narrower size distribution than the Pari (air-jet) nebuliser. Unlike the vibrating-mesh nebulisers, the performance of the jet nebuliser was largely independent of formulation. A nebuliser-dependent significant loss of the originally entrapped drug was demonstrated. A customised large mesh Aeroneb Pro reduced the drug losses during nebulisation. High sensitivity differential scanning calorimetry showed that the phospholipid phase transitions and liposomal bilayer interaction with beclometasone dipropionate were dependent on the method of liposome manufacture. Ethanol-based proliposomes produced liposomes having no pretransition, with a very low incorporation of the steroid (max. 1 mole%). This was attributed to an alcohol-induced interdigitation of the bilayers. 1 to 2.5 mole% steroid seemed to be optimal for incorporation in liposomes manufactured by the thin film or particulate-based proliposome method. Jet-nebulisation of particulate-based proliposomes delivered vesicles with enhanced steroid incorporation compared to liposomes generated by manual dispersion of these proliposomes

Characterization and manipulation of lipid self-assembly to construct stable, portable synthetic lipid bilayers

The overarching goal of this research work is to further our understanding of lipid self-assembly and its organization at an oil-water interface to support the development of synthetic lipid bilayer systems that can be used in biologically relevant fields such as membrane biophysics, protein electrophysiology, development of synthetic biomolecules, drugs, nanoparticles and other applications. Self-assembly kinetics and interfacial properties of lipid monolayers formed at a liquid-air and liquid-liquid interface are characterized using Langmuir-Blodgett trough and pendant drop tensiometer. Insights gained from these studies not only allow us to answer questions related to droplet interface bilayer (DIB; a promising technique to assemble artificial lipid membranes) formation but also enable us to manipulate properties of monolayer in order to improve the potential of droplet interface bilayer by, a) increasing the number of phospholipids that can form DIBs, b) improving the success rate of DIB formation, and c) enhancing the electrical stability of bilayers formed. Owing to its wide range of applicability, novel efforts towards improving the durability and portability of DIB system are presented. In addition, this research work aims at using Nanoscribe direct laser writing — a state-of-the-art 3D printing device, to build 3D micro-scaffolds that can support lipid monolayers and bilayers that are suitable for high resolution optical studies

Electrodynamic droplet actuation for lab on a chip system

This work presents the development of electrowetting on dielectric and liquid dielectrophoresis as a platform for chemistry, biochemistry and biophysics. These techniques, typically performed on a single planar surface offer flexibility for interfacing with liquid handling instruments and performing biological experimentation with easy access for visualisation. Technology for manipulating and mixing small volumes of liquid in microfluidic devices is also crucially important in chemical and biological protocols and Lab on a Chip devices and systems. The electrodynamic techniques developed here have rapid droplet translation speeds and bring small droplets into contact where inertial dynamics achieve rapid mixing upon coalescence.In this work materials and fabrication processes for both electrowetting on dielectric and liquid dielectrophoresis technology have been developed and refined. The frequency, voltage and contact angle dependent behaviour of both techniques have been measured using two parallel coplanar electrodes. The frequency dependencies of electrowetting and dielectrophoretic liquid actuation indicate that these effects are high and low-frequency limits, respectively, of a complex set of forces. An electrowetting based particle mixer was developed using a custom made electrode array and the effect of varying voltage and frequency on droplet mixing was examined, with the highest efficiency mixing being achieved at 1 kHz and 110 V in about 0.55 seconds.A composite electrodynamic technique was used to develop a reliable method for the formation of artificial lipid bilayers within microfluidic platforms for measuring basic biophysical aspects of cell membranes, for biosensing and drug discovery applications. Formation of artificial bilayer lipid membranes (BLMs) was demonstrated at the interface of aqueous droplets submerged in an organic solvent-lipid phase using the liquid dielectrophoresis methods developed in this project to control the droplet movement and bring multiple droplets into contact without coalescence. This technique provides a flexible, reconfigurable method for forming, disassembling and reforming BLMs within a microsystem under simple electronic control. BLM formation was shown to be extremely reliable and the BLMs formed were stable (with lifetimes of up to 20 hours) and therefore were suitable for electrophysiological analysis. This system was used to assess whether nanoparticle-membrane contact leads to perturbation of the membrane structure. The conductance of artificial membranes was monitored following exposure to nanoparticles using this droplet BLM system. It was demonstrated that the presence of nanoparticles with diameters between 50 and 500 nm can damage protein-free membranes at particle concentrations in the femtomolar range. The effects of particle size and surface chemistry were also investigated. It was shown that a large number of nanoparticles can translocate across a membrane, even when the surface coverage is relatively low, indicating that nanoparticles can exhibit significant cytotoxic effects

Microfluidic Planar Phospholipids Membrane System Advancing Dynamics Studies of Ion Channels and Membrane Physics

The interrogation of lipid membrane and biological ion channels supported within bilayer phospholipid membranes has greatly expanded our understanding of the roles membrane and ion channels play in a host of biological functions. Several key drawbacks of traditional electrophysiology systems used in these studies have long limited our effort to study the ion channels. Firstly, the large volume buffer in this system typically only allows single or multiple additions of reagents, while complete removal either is impossible or requires tedious effort to ensure the stability of membrane. Thus, it has been highly desirable to be able to rapidly and dynamically modulate the (bio)chemical conditions at the membrane site. Second, it is difficult to change temperature effectively with large thermal mass in macro device. Third, traditional PPM device host vertical membranes, therefore incompatible with confocal microscopy techniques. The miniaturization of bilayer phospholipid membrane has shown potential solution to the drawbacks stated above. A simple microfluidic design is developed to enable effective and robust dynamic perfusion of reagents directly to an on-chip planar phospholipid membrane (PPM). It allows ion channel conductance to be readily monitored under different dynamic reagent conditions, with perfusion rates up to 20 µL/min feasible without compromising the membrane integrity. It is estimated that the lower limit of time constant of kinetics that can be resolved by our system is 1 minute. Using this platform, the time-dependent responses of membrane-bound ceramide ion channels to treatments with La3+ and a Bcl-xL mutant were studied and the results were interpreted with a novel elastic biconcave distortion model. Another engineering challenge this dissertation takes on is the integration of fluorescence studies to micro-PPM system. The resulting novel microfluidic system enables high resolution, high magnification and real-time confocal microscope imaging with precise top and bottom (bio)chemical boundary conditions defined by perfusion, by integrating in situ PPM formation method, perfusion capability and microscopy compatibility. To demonstrate such electro-optical chip, lipid micro domains were imaged and quantitatively studied for their movements and responses to different physical parameters. As an extension to this platform, a double PPM system has been developed with the aim to study interactions between two membranes. Potential application in biophysics and biochemistry using those two platforms were discussed. Another important advantage of microfluidics is its lower thermal mass and compatibility with various microfabrication methods which enables potential integration of local temperature controller and sensor. A prototype thermal PPM chip is also discussed together with some preliminary results and their implication on ceramide channel assembly and disassembly mechanism

Bio-Inspired Nanomembranes as Building Blocks for Nanophotonics, Plasmonics and Metamaterials

Nanomembranes are the most widespread building block of life, as they encompass cell and organelle walls. Their synthetic counterparts can be described as freestanding or free-floating structures thinner than 100 nm, down to monatomic/monomolecular thickness and with giant lateral aspect ratios. The structural confinement to quasi-2D sheets causes a multitude of unexpected and often counterintuitive properties. This has resulted in synthetic nanomembranes transiting from a mere scientific curiosity to a position where novel applications are emerging at an ever-accelerating pace. Among wide fields where their use has proven itself most fruitful are nano-optics and nanophotonics. However, the authors are unaware of a review covering the nanomembrane use in these important fields. Here, we present an attempt to survey the state of the art of nanomembranes in nanophotonics, including photonic crystals, plasmonics, metasurfaces, and nanoantennas, with an accent on some advancements that appeared within the last few years. Unlimited by the Nature toolbox, we can utilize a practically infinite number of available materials and methods and reach numerous properties not met in biological membranes. Thus, nanomembranes in nano-optics can be described as real metastructures, exceeding the known materials and opening pathways to a wide variety of novel functionalities

Chemical Science Membrane protein biosensing with plasmonic nanopore arrays and pore-spanning lipid membranes †

Integration of solid-state biosensors and lipid bilayer membranes is important for membrane protein research and drug discovery. In these sensors, it is critical that the solid-state sensing material does not have adverse effects on the conformation or functionality of membrane-bound molecules. In this work, pore-spanning lipid membranes are formed over an array of periodic nanopores in free-standing gold films for surface plasmon resonance (SPR) kinetic binding assays. The ability to perform kinetic assays with a transmembrane protein is demonstrated with a-hemolysin (a-HL). The incorporation of a-HL into the membrane followed by specific antibody binding (anti-a-HL) red-shifts the plasmon resonance of the gold nanopore array, which is optically monitored in real time. Subsequent fluorescence imaging reveals that the antibodies primarily bind in nanopore regions, indicating that a-HL incorporation preferentially occurs into areas of pore-spanning lipid membranes