Ionic-type transistors are important devices for precise chemical control and biosensing applications. Previous work by Tybrandt et al. has demonstrated a novel approach to constructing an ionic transistor using conducting polymers poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and quarternized- polyvinyl benzyl chloride (q-PVBC). This approach could be combined with the 3D stamp method of generating concentration gradients in supported lipid bilayers (SLBs) as shown by Liu et al. to create a charged lipid-based ionic polar junction transistor. An electric potential applied across the SLB would drive charged lipids towards the opposite electrode, thus generating current flow across the SLB. Incorporation of a charged-lipid functionalized PEDOT derivative as demonstrated by Johansson et al. would allow a longer period of current flow before charge carriers are depleted. Such a device could offer novel approaches to biosensing.

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Membranes are essential components of all living organisms forming the borders of cells and their organelles. Planar lipid membranes deposited on solid substrates (solid supported membranes) provide models to study the functions of membrane proteins and are used as biosensing platforms. However, despite remarkable progress, solid supported membranes are not stable to harsh conditions such as dehydration, high temperature and pressure, and mechanical stress. In addition, the direct deposition of membranes onto a solid substrate often causes restricted mobility and denaturation of reconstituted membrane proteins. Membrane stability can be addressed by altering the structure of the component lipids. Bolalipids are an interesting class of bipolar lipids that have been proposed for biosensing applications. Membranes formed from mixtures of a bolalipid, C20BAS, and dioleoylphosphaphatidylcholine, POPC, were characterized by atomic force spectroscopy (AFM). The lipid mixtures produced a phase separated membrane consisting of thinner bolalipid-rich and thicker monopolar-rich POPC regions, with a height difference of approximately 1-2 nm. This confirmed an earlier prediction that some bolalipid/PC membranes would phase separate due to the hydrophobic mismatch between the two lipids. Interestingly, the surface coverage of the two phases was inconsistent with what one would expect from the initial starting lipid ratios. The complex membrane morphologies observed were accredited to the interplay of several factors, including a compositionally heterogeneous vesicle population, exchange of lipid between the vesicle solution and solid substrate during formation of the supported membrane, and slow equilibration of domains due to pinning of the lipids to the solid support. Decoupling the membrane from its underlying surface is one strategy to maintain the structure and mobility of membrane proteins. This decoupling can be achieved by depositing the membrane on a soft cushion composed of a water swelling hydrophilic polymer. A polyelectrolyte multilayer (PEM) and a tethered poly(ethylene) glycol (PEG) polymer are the two types of polymer cushions used in this study. The PEMs consist of the charged polysaccharides, chitosan (CHI) and hyaluronic acid (HA) which offer the advantage of biocompatibility over synthetic PEMs. DOPC lipid bilayers were formed at pH 4 and 6.5 on (CHI/HA)5 films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; fluorescence and AFM showed that this was accredited to the formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. However, more uniform bilayers with mobile lipids were produced at pH 4. Measured diffusion coefficients were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. The suitability of polymer supported membranes for the incorporation of integral membrane proteins was also assessed. The integral membrane protein Ste14p, a 26 kDa methyltransferase enzyme, was reconstituted into POPC membranes on PEM and PEG supports. A combination of fluorescence microscopy, FRAP, AFM and an in situ methyltransferase activity assay were utilized to characterize the protein incorporated polymer supported membranes. Fluorescence measurements showed that more protein was incorporated in model membranes formed on the PEG support, compared to either glass or PEM cushions. However, the protein activity on a PEG support was comparable to that of the protein in a membrane on glass. FRAP measurements showed that the lipid mobilities of the POPC:Ste14p bilayers on the various supports were also comparable. Lastly, as a new platform for manipulating and handling membrane proteins, nanodiscs containing reconstituted Ste14p were studied. Nanodiscs are small, soluble and stable bilayer discs that permit the study of membrane proteins in a uniform phospholipid bilayer environment. Empty and protein containing nanodiscs were deposited on a mica surface and imaged by AFM. AFM showed that protein containing samples possessed two subpopulations of nanodiscs with a height difference of ~1 nm. The taller discs, ~20% of the population, contained protein. Other experiments showed that the packing of the nanodisc samples was influenced by their initial stock concentration and that both imaging force and the addition of Mg2+ caused formation of larger bilayer patches.

Understanding the cellular membrane interaction with membrane active biomolecules and antimicrobial agents provides an insight in their working mechanism. Here, we studied the effect of antimicrobial agents; a recently developed peptidomimetics E107-3 and colistin as well as the N-terminal helix H0, of Endophilin A1 on the lipid bilayer. It is important to discern the interaction mechanism of antimicrobial peptides with lipid membranes in battling multidrug resistant bacterial pathogens. We study the modification of structural and mechanical properties with a recently reported peptidomimetic on lipid bilayer. The compound referred to as E107-3 is synthesized based on the acylated reduced amide scaffold and has been shown to exhibit good antimicrobial potency. This compound increases lipid bilayer permeability as indicated by our vesicle leakage essay. Micropipette aspiration experiment shows that exposure of GUV to the compound causes the protrusion length Lp to spontaneously increase and then decrease, followed by GUV rupture. Solution atomic force microscopy (AFM) is used to visualize lipid bilayer structural modulation within a nanoscopic regime. This compound induces nanoscopic heterogeneous structures rather than pore like structures as produced by melittin. Finally, we use AFM-based force spectroscopy to study the impact of the compound on lipid bilayer’s mechanical properties. With the incremental addition of this compound, we found the bilayer puncture force decreases moderately and a 39% decrease of the bilayer area compressibility modulus KA. To explain our experimental data, we propose a membrane interaction model encompassing disruption of lipid chain packing and extraction of lipid molecules. The later action mode is supported by our observation of a double-bilayer structure in the presence of fusogenic calcium ions. Polyanionic Lipopolysaccharides LPS are important in regulating the permeability of outer membrane (OM) of gram-negative bacteria. To initiate the bactericidal activity of polymyxins, it is essential to impair the LPS-enriched OM. Here, we study the mechanism of membrane permeability caused by colistin (Polymyxin E) of LPS/phospholipid bilayers. Our vesicle leakage experiment showed that colistin binding enhanced bilayer permeability; the maximum increase in the bilayer permeability was positively correlated with the LPS fraction. Addition of magnesium ions abolished the effect of LPS in enhancing bilayer permeabilization. Solution atomic force microscopy (AFM) measurements on planar lipid bilayers shows the formation of nano- and macro clusters which protruded from the bilayer by ~2nm. Moreover, increasing the fraction of LPS or colistin enhances the formation of clusters but inhibits by magnesium ions addition. To explain our experimental data, we proposed a lipid-clustering model where colistin binds to LPS to form large-scale complexes segregated from zwitterionic phospholipids. The discontinuity (and thickness mismatch) at the edge of LPS-colistin clusters will create a passage that allows solutes to permeate through. The proposed model is consistent with all data obtained from our leakage and AFM experiments. Our results of LPS-dependent membrane restructuring provided useful insights into the mechanism that could be used by polymyxins in impairing the permeability barrier of the OM of Gram-negative bacteria. Also, we studied the effect of helix H0 of a membrane modification inducing protein endophilin, on planar bilayer. We obtained transmembrane defects on the bilayer when scanned.with AFM.

Indiana University-Purdue University Indianapolis (IUPUI) / The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight. Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction. To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18596号 / 工博第3957号 / 新制||工||1608(附属図書館) / 31496 / 京都大学大学院工学研究科化学工学専攻 / (主査)教授 山本 量一, 教授 秋吉 一成, 准教授 谷口 貴志, 教授 大嶋 正裕 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

This dissertation presents the development of a modeling scheme that is developed to model the membrane potentials and ion currents through a bilayer network system. The modeling platform builds off of work performed by Hodgkin and Huxley in modeling cell membrane potentials and ion currents with electrical circuits. This modeling platform is built specifically for cell mimics where individual aqueous volumes are separated by single bilayers like the droplet-interface-bilayer. Applied potentials in one of the aqueous volumes will propagate through the system creating membrane potentials across the bilayers of the system and ion currents through the membranes when proteins are incorporated to form pores or channels within the bilayers. The model design allows the system to be divided into individual nodes of single bilayers. The conductance properties of the proteins embedded within these bilayers are modeled and a finite element analysis scheme is used to form the system equations for all of the nodes. The system equation can be solved for the membrane potentials through the network and then solve for the ion currents through individual membranes in the system. A major part of this work is modeling the conductance of the proteins embedded within the bilayers. Some proteins embedded in bilayers open pores and channels through the bilayer in response to specific stimuli and allow ion currents to flow from one aqueous volume to an adjacent volume. Modeling examples of the conductance behavior of specific proteins are presented. The examples demonstrate aggregate conductance behavior of multiple embedded proteins in a single bilayer, and at examples where few proteins are embedded in the bilayer and the conductance comes from a single-channel or pore. The effect of ion gradients on the single channel conductance example is explored and those effects are included in the single-channel conductance model. Ultimately these conductance models are used with the system model to predict ion currents through a bilayer or through part of a bilayer network system. These modeling efforts provide a modeling tool that will assist engineers in designing bilayer network systems. / Ph. D.

This dissertation presents the development of a new form of biomolecular material system which features interface lipid bilayers capable of hosting a wide variety of natural and engineered proteins. This research builds on the droplet interface bilayer (DIB) platform which first demonstrated that, through self-assembly, lipid-encased water droplets submersed in oil can be physically connected to form a liquid-supported lipid bilayer at the droplet interface. Key advantages of the DIB method over previous bilayer formation techniques include the lack of a supporting substrate which simplifies bilayer formation and the ability to connect many droplets to form `cell-inspired' networks which can provide a collective utility based on the compositions and arrangement of the droplets. The research present herein specifically seeks to overcome three limitations of the original droplet interface bilayer: limited portability due to lack of droplet support, the use of externally supported electrodes to electrically probe the network, and the requirement that in order to form DIB networks, aqueous volumes must be individually dispensed and arranged. The approach presented in this document is to provide increased interactions between the contained liquid phases and a supporting substrate in order to achieve both increased usability through refined methods of packaging and in situ interface formation which eliminates the need to create individual droplets. Physical encapsulation is defined as the the use of a solid substrate to contain both liquid phases such that the aqueous volumes are physically supported on one length scale (10-1000µm) while not inhibiting the self-assembly of phospholipids at the oil/water interface occurring on a much smaller length scale (1-10nm). Physically-encapsulated droplet interface bilayers are achieved by connecting lipid-encased droplets within a substrate that tightly confines the positions of neighboring droplets. A term called the packing factor is introduced to quantify the ratio of the aqueous volumes per the total compartment volume. Physically-encapsulated droplet interface bilayers formed in high packing factor substrate (30%) that also features integrated electrodes demonstrate all of the properties that unencapsulated DIBs exhibit (electrical resistances greater than 1GΩ, failure potentials between |200-300|mV, and the ability to host transmembrane proteins) but these confined assemblies can be moved, shaken, and even completely inverted. Additionally, a structured experiment to quantify the durability of interface bilayers shows that encapsulated and unencapsulated droplet interface bilayers can both survive 3-7g of lateral acceleration prior to bilayer failure, but have different modes of failure. Encapsulated DIBs tend to rupture, while unencapsulated DIBs completely separate. Physical encapsulation is also shown to permit the in situ formation of durable interface bilayers when the substrate is made from a flexible material. The importance of this approach stems from the fact that, by using the substrate to locally partition a single aqueous volume into multiple volumes, there is no need to arrange individual droplets. This method of bilayer formation is termed the regulated attachment method (RAM), since the separation and subsequent reattachment of the aqueous volumes is regulated by the opening and closing of an aperture within the flexible substrate. In this dissertation, a mechanical force is used to directly modulate the aperture dimension for controlling both the initial formation and final size of the interface. With the demonstrated advantages of portability and controlled attachment offered by physical encapsulation, encapsulated lipid bilayers are formed within a completely sealed flexible substrate. A key aspect of this final work is to demonstrate that both the organic and aqueous phases can be stabilized internally, creating a complete material system that features tailorable interface bilayers. / Ph. D.

Anyone who has eaten spicy foods has experienced the adverse effects of capsaicin, the pungent chemical found in hot chili that causes a burning sensation. The specific action of capsaicin occurs by the activation of receptors in sensory neurons. This thesis investigates the interaction of capsaicin with model cell membranes representing the structure of neurons. In particular, we are interested in the changes induced by capsaicin to the structure and dynamics of membranes. Molecular dynamics simulations are used to study the molecular interactions. The first part of this study evaluates different molecular representations for capsaicin in an 1-octanol/water system. This inhomogeneous system is commonly used to determine the partition of compounds between hydrophilic and hydrophobic environments, as that found in biological membranes. The results of these simulations validate the OPLS united-atom force field as a reasonable molecular representation of capsaicin, as it describes the behavior of capsaicin both quantitatively and qualitatively in 1-octanol/water mixtures. In the second part, simulations are performed for capsaicin and model cell membranes consisting of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine, two of the most commonly found lipids. Simulations investigated capsaicin in the aqueous and lipid phases. The results provide insight into the changes to the bilayers caused by capsaicin. Bilayers containing dipalmitoylphosphatidylethanolamine showed lower permeabilities to capsaicin than those composed of pure dipalmitoylphosphatidylcholine. Temperature is found to be an important factor in the permeability of capsaicin in the bilayer. Capsaicin in the bilayer concentrated in a region beneath the lipid/water interface, in which favorable hydrophilic and lipophilic interactions occur. The structure of the bilayer is not significantly changed at the concentrations of capsaicin considered. One important result from the simulations indicates that the interfacial density decreases with increasing capsaicin concentration in the bilayer, supporting the experimental observations of increased permeability in bilayers exposed to capsaicin. / Master of Science

This dissertation investigates the behavior of the Escherichia Coli mechanosensitive (MS) channel MscL, when incorporated within a droplet interface bilayer (DIB). The activity of MscL channels in an artificial DIB system is demonstrated for the first time in this document. The DIB represents a building block whose repetition can form the basis to a new class of smart materials. The corresponding stimuli-responsive properties can be controlled by the type of biomolecule incorporated into the lipid bilayer, which is in the heart of this material. In the past decade, many research groups have proven the capability of the DIB to host a wide collection of natural and engineered functional biomolecules. However, very little is known about the mechano-electrical transduction capabilities of the DIB. The research present herein specifically seeks to achieve three direct goals: 1) exploring the capabilities of the DIB to serve as a platform for mechano-electrical transduction through the incorporation of bacterial MscL channels, 2) understanding the physics of mechano-electrical transduction in the DIB through the development of theoretical models, and 3) using the developed science to regulate the response of the DIB to a mechanical stimulus. MscL channels, widely known as osmolyte release valves and fundamental elements of the bacterial cytoplasmic membrane, react to increased tension in the membrane. In the event of hypo-osmotic shocks, several channels residing in the membrane of a small cell can generate a massive permeability response to quickly release ions and small molecules, saving bacteria from lysis. Biophysically, MscL is well studied and characterized primarily through the prominent patch clamp technique. Reliable structural models explaining MscL's gating mechanism are proposed based on its homolog's crystal structure modeling, which lead to extensive experimentation. Under an applied tension of ~10 mN/m, the closed channel which consists of a tight bundle of transmembrane helices, transforms into a ring of greatly tilted helices forming an ~8 A water-filled conductive pore. It has also been established that the hydrophobicity of the tight gate, positioned at the intersection of the inner TM1 domains, determines the activation threshold of the channel. Correspondingly, it was found that by decreasing the hydrophobicity of the gate, the tension threshold could be lowered. This property of MscL made possible the design of various controllable valves, primarily for drug delivery purposes. For all the aforementioned properties and based on its fundamental role of translating cell membrane excessive tensions into electrophysiological activities, MscL makes a great fit as a mechanoelectrical transducer in DIBs. The approach presented in this document consists of increasing the tension in the lipid bilayer interface through the application of a dynamic mechanical stimulus. Therefore, a novel and simple experimental apparatus is assembled on an inverted microscope, consisting of two micropipettes (filled with PEG-DMA hydrogel) containing Ag/AgCl wires, a cylindrical oil reservoir glued on top of a thin acrylic sheet, and a piezoelectric oscillator actuator. By using this technique, dynamic tension can be applied by oscillating one droplet, producing deformation of both droplets and area changes of the DIB interface. The tension in the artificial membrane will cause the MS channels to gate, resulting in an increase in the conductance levels of the membrane. The increase in bilayer tension is found to be equal to the sum of increase in tensions in both contributing monolayers. Tension increase in the monolayers occurs due to an increase in surface area of the constant volume aqueous droplets supporting the bilayer. The results show that MS channels are able to gate under an applied dynamic tension. Interestingly, this work has demonstrated that both electrical potential and surface tension need to be controlled to initiate mechanoelectric coupling, a property previously not known for ion channels of this type. Gating events occur consistently at the peak compression, where the tension in the bilayer is maximal. In addition, the experiments show that no activity occurred at low amplitude oscillations (< 62.5um). These two findings basically present an initial proof that gating is occurring and is due to the mechanical excitation, not just a random artifact. The role of the applied potential is also highlighted in this study, where the results show that no gating happens at potentials lower that 80 mV. The third important observation is that the frequency of oscillation has an important impact of the gating probability, where no gating is seen at frequencies higher than 1 Hz or lower than 0.1 Hz. Each of the previous observations is addressed separately in this research. It was found that the range of frequencies to which MscL would respond to in a DIB could be widened by using asymmetrical sinusoidal signals to stimulate the droplets. By increasing the relaxation time and shorting the compression time, a change in the monolayer's surface area is achieved, thus higher tension increase in the bilayer. It was also found that a high membrane potential assists in the opening of MscL as the droplets are stimulated. This is due to the sensitivity of MscL to the polarity of the signal. By using the right polarity the channel could be regulated to become more susceptible to opening, even at tensions lower than the threshold. Finally, it was demonstrated, for the first time, that MscL would gate in asymmetric bilayers without the need to apply a high external potential. Asymmetric bilayers, which are usually composed from different lipids in each leaflet, generate an asymmetric potential at the membrane. This asymmetric potential is proven to be enough to cause MscL to gate in DIBs upon stimulation. / Ph. D.