Publications

2017   >>

The response of lipid bilayers to osmotic stress is an important part of cellular function. Recent ex5 perimental studies showed that when cell-sized giant unilamellar vesicles (GUVs) are exposed to hypotonic media, they respond to the osmotic assault by undergoing a cyclical sequence of swelling and bursting events, coupled to the membrane’s compositional degrees of freedom. Here, we establish a fundamental and quantitative under8 standing of the essential pulsatile behavior of GUVs under hypotonic conditions by advancing a comprehensive theoretical model of vesicle dynamics. The model quantitatively captures the experimentally measured swell10 burst parameters for single-component GUVs, and reveals that thermal fluctuations enable rate-dependent pore nucleation, driving the dynamics of the swell-burst cycles. We further extract constitutional scaling relationships between the pulsatile dynamics and GUV properties over multiple time scales. Our findings provide a fundamental framework that has the potential to guide future investigations on the non-equilibrium dynamics of vesicles under osmotic stress.

DOI:10.1016/j.bpj.2017.03.018

The goal of this pilot study was to determine whether HDL glycoprotein composition affects HDL’s immunomodulatory function. HDL were purified from healthy controls (n = 13), subjects with metabolic syndrome (MetS) (n = 13), and diabetic hemodialysis (HD) patients (n = 24). Concentrations of HDL-bound serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), apolipoprotein A-I (ApoA-I), apolipoprotein C-III (ApoC-III), α-1-antitrypsin (A1AT), and α-2-HS-glycoprotein (A2HSG); and the site-specific glycovariations of ApoC-III, A1AT, and A2HSG were measured. Secretion of interleukin 6 (IL-6) in lipopolysaccharide-stimulated monocytes was used as a prototypical assay of HDL’s immunomodulatory capacity. HDL from HD patients were enriched in SAA, LBP, ApoC-III, di-sialylated ApoC-III (ApoC-III2) and desialylated A2HSG. HDL that increased IL-6 secretion were enriched in ApoC-III, di-sialylated glycans at multiple A1AT glycosylation sites and desialylated A2HSG, and depleted in mono-sialylated ApoC-III (ApoC-III1). Subgroup analysis on HD patients who experienced an infectious hospitalization event within 60 days (HD+) (n = 12), vs. those with no event (HD−) (n = 12) showed that HDL from HD+ patients were enriched in SAA but had lower levels of sialylation across glycoproteins. Our results demonstrate that HDL glycoprotein composition, including the site-specific glycosylation, differentiate between clinical groups, correlate with HDL’s immunomodulatory capacity, and may be predictive of HDL’s ability to protect from infection.

DOI:10.1038/srep43728

Many common amphiphiles self-assemble in water to produce heterogeneous populations of discrete and symmetric but polydisperse and multilamellar vesicles isolating the encapsulated aqueous core from the surrounding bulk. But when mixtures of amphiphiles of vastly different elastic properties co-assemble, their non-uniform molecular organization can stabilize lower symmetries and produce novel shapes. Here, using high resolution electron cryomicroscopy and tomography, we identify the spontaneous formation of a membrane morphology consisting of unilamellar tubular vesicles in dilute aqueous solution of binary mixtures of two different amphiphiles of vastly different origins. Our results show that aqueous phase mixtures of a fluid-phase phospholipid and an amphiphilic block copolymer spontaneously assume a bimodal polymorphic character in a composition dependent manner: over a broad range of compositions (15-85 mol% polymer component), a tubular morphology co-exists with spherical vesicles. Strikingly, in the vicinity of equimolar compositions, an exclusively tubular morphology (Lt; diameter, ~15 nm; length, > 1 µm; core, ~ 2.0 nm; wall, ~ 5-6 nm) emerges in an apparent steady state. Theory suggests that the spontaneous stabilization of cylindrical vesicles, unaided by extraneous forces, requires a significant spontaneous bilayer curvature, which in turn necessitates a strongly asymmetric membrane composition. We confirm that such dramatic compositional asymmetry is indeed produced spontaneously in aqueous mixtures of a lipid and polymer through two independent biochemical assays – (1) reduction in the quenching of fluorophore-labeled lipids and (2) inhibition in the activity of externally added lipid-hydrolyzing phospholipase A2 establishing a significant enrichment of the polymer component in the outer leaflet. Taken together, these results illustrate the coupling of membrane shape with local composition through spontaneous curvature generation under conditions of asymmetric distribution of mixtures of disparate amphiphiles.

DOI:10.1039/C6SM01753C

2016   >>

Lysosomotropic detergents (LDs) selectively rupture lysosomal membranes through mechanisms that have yet to be characterized. A consensus view, currently, holds that LDs, which are weakly basic, diffuse across cellular membranes as monomers in an uncharged state, and via protonation in the acidic lysosomal compartment, they become trapped and accumulate, and subsequently solubilize the membrane and induce lysosomal membrane permeabilization. Here, we demonstrate that the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) spontaneously assembles into vesicles at, and above, cytosolic pH, and that the vesicles disassemble as pH reaches 6.4 or lower. The aggregation commences at concentrations below the range of those used in cell studies. Assembly and disassembly of the vesicles was studied via dynamic light scattering, zeta potential measurements, cryo-TEM and fluorescence correlation spectroscopy, and was found to be reversible via control of the pH. Aggregation of MSDH into closed vesicles under cytosolic conditions is at variance with the commonly held view of LD behaviour, and we propose that endocytotic pathways should be considered as possible routes of LD entry into lysosomes. We further demonstrate that MSDH vesicles can be loaded with fluorophores via a solution transition from low to high pH, for subsequent release when the pH is lowered again. The ability to encapsulate molecular cargo into MSDH vesicles together with its ability to disaggregate at low pH and to permeabilize the lysosomal membrane presents an intriguing possibility to use MSDH as a delivery system.

DOI:10.1021/acs.langmuir.6b03458

Using single-particle tracking, we investigate the interaction of small unilamellar vesicles (SUVs) that are electrostatically tethered to the freestanding membrane of a giant unilamellar vesicle (GUV). We find that the surface mobility of the GUV-riding SUVs is Brownian, insensitive to the bulk viscosity, vesicle size, and vesicle fluidity but strongly altered by the viscosity of the underlying membrane. Analyzing the diffusional behavior of SUVs within the Saffman–Delbrück model for the dynamics of membrane inclusions supports the notion that the mobility of the small vesicles is coupled to that of dynamically induced lipid clusters within the target GUV membrane. The reversible binding also offers a nonperturbative means for measuring the viscosity of biomembranes, which is an important parameter in cell physiology and function.

DOI:10.1021/acs.langmuir.6b00985

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.

DOI:10.1038/srep26823

The cholesterol partitioning and condensing effect in the liquid-ordered (Lo) and liquid-disordered (Ld) phases were systematically investigated for ternary mixture lipid multilayers consisting of 1:1 1,2-dipalmitoyl -sn-glycero-3-phosphocholine /1,2-dioleoyl-sn-glycero-3- phosphocholine with varying concentrations of cholesterol. X-ray lamellar diffraction was used to deduce the electron density profiles of each phase. The cholesterol concentration in each phase was quantified by fitting of the electron density profiles with a newly invented basic lipid profile scaling method that minimizes the number of fitting parameters. The obtained cholesterol concentration in each phase versus total cholesterol concentration in the sample increases linearly for both phases. The condensing effect of cholesterol in ternary lipid mixtures was evaluated in terms of phosphate-to-phosphate distances, which together with the estimated cholesterol concentration in each phase was converted into an average area per molecule. In addition, the cholesterol position was determined to a precision of (±0.7Å) and an increase of disorder in the lipid packing in the Lo phase was observed for total cholesterol concentration of 20∼30%.

DOI:10.1016/j.bpj.2016.02.022

Giant lipid vesicles are topologically closed compartments bounded by semipermeable flexible shells, which isolate femto- to picoliter quantities of the aqueous core from the surrounding bulk. Although water equilibrates readily across vesicular walls (10^–2–10^–3 cm^3 cm^–2 s^–1), the passive permeation of solutes is strongly hindered. Furthermore, because of their large volume compressibility (∼10^9–10^10 N m^–2) and area expansion (10^2–10^3 mN m^–1) moduli, coupled with low bending rigidities (10^–19 N m), vesicular shells bend readily but resist volume compression and tolerate only a limited area expansion (∼5%). Consequently, vesicles experiencing solute concentration gradients dissipate the available chemical energy through the osmotic movement of water, producing dramatic shape transformations driven by surface-area–volume changes and sustained by the incompressibility of water and the flexible membrane interface. Upon immersion in a hypertonic bath, an increased surface-area–volume ratio promotes large-scale morphological remodeling, reducing symmetry and stabilizing unusual shapes determined, at equilibrium, by the minimal bending-energy configurations. By contrast, when subjected to a hypotonic bath, walls of giant vesicles lose their thermal undulation, accumulate mechanical tension, and, beyond a threshold swelling, exhibit remarkable oscillatory swell–burst cycles, with the latter characterized by damped, periodic oscillations in vesicle size, membrane tension, and phase behavior. This cyclical pattern of the osmotic influx of water, pressure, membrane tension, pore formation, and solute efflux suggests quasi-homeostatic self-regulatory behavior allowing vesicular compartments produced from simple molecular components, namely, water, osmolytes, and lipids, to sense and regulate their microenvironment in a negative feedback loop.

DOI: 10.1021/acs.langmuir.5b04470

The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of ∼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity.

DOI:j.bpj.2015.11.032

2015

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

DOI: 10.1038/srep16432

A fluid lipid bilayer in water fluctuates freely. Even in the absence of specific chemical interactions, a complex interplay of a variety of nonspecific forces—attractive and repulsive, short- and long-ranged—determine the equilibrium separation between these well-hydrated bilayers (1). It also plays critical roles in many biological processes, such as cell adhesion and membrane fusion, in which these surfaces are pushed closer together. Contributing to the interplay are coupled influences of the classical DLVO (named after Derjaguin, Landau, Verwey, and Overbeek, who described forces between small, smooth, and charged surfaces in water) and non-DLVO forces. These forces include a) the omnipresent van der Waals force, which provides a weak attraction in a relatively long-ranged manner, and b) the electrostatic double-layer forces between charged membranes. The non-DLVO forces, contributing to interbilayer interactions, include c) the so-called hydration force (a short-range, exponentially decaying repulsive interaction), which is thought to originate from surface-induced perturbation of water dipoles and its propagation away from the interface through water-water interactions (2), and d) the long-range repulsive Helfrich force, which arises from the suppression of the free fluctuations of single bilayers, resulting in entropic loss when two membranes come closer together (3). While the first three direct molecular forces (a-c) can be treated independently and additively, the contributions from the fluctuation-induced Helfrich forces (d) are not readily separable, and couple to the other forces in subtle and complex ways.

DOI: 10.1016/j.bpj.2015.05.008

We report a new and simple approach to prepare a class of silica-reinforced liposomes with hybrid core-shell nanostructures. The amphiphilic natural structure of lipids was exploited to sequester hydrophobic molecules, namely precursor TEOS and pyrene, in the hydrophobic mid-plane of liposomal bilayer assemblies in the aqueous phase. Subsequent interfacial hydrolysis of TEOS at the bilayer/water interface and ensuing condensation within the hydrophobic interstices of the lipid bilayer drives silica formation in-situ, producing a novel class of silica-lipid hybrid liposils. Structural characterization by scanning- and transmission electron microscopy confirm that the liposils so generated preserve closed topologies and size-monodipersity of the parent lecithin liposomes, and DSC-TGA and XRD measurements provide evidence for the silica coating. Monitoring fluorescence measurements using embedded pyrene yield detailed information of microenvironment changes, which occur during sol-gel process and shed light on the structural evolution during silica formation. We envisage that liposils formed by this simple, new approach, exploiting the hydrophobic core of the lipid bilayer to spatially localize silica-forming precursors enables preparation of stable liposils exhibiting capacity for cargo encapsulation, bicompatibility, and fluorescence monitoring, more generally opening a window for construction of stable, functional hybrid materials.

DOI: 10.1021/acsami.5b01386

During vesicle budding or endocytosis, biomembranes undergo a series of lipid- and protein-mediated deformations involving cholesterol-enriched lipid rafts. If lipid rafts of high bending rigidities become confined to the incipient curved membrane topology such as a bud-neck interface, they can be expected to reform as ring-shaped rafts. Here, we report on the observation of a disk-to-ring shape morpho-chemical transition of a model membrane in the absence of geometric constraints. The raft shape transition is triggered by lateral compositional heterogeneity and is accompanied by membrane deformation in the vertical direction, which is detected by height-sensitive fluorescence interference contrast microscopy. Our results suggest that a flat membrane can become curved simply by dynamic changes in local chemical composition and shape transformation of cholesterol-rich domains.

DOI: 10.1021/jacs.5b04559

Through attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), we have studied the adsorption characteristics of small unilamellar vesicles (SUVs) of a gel-phase phospholipid, dipalmitoylphosphatidylcholine (DPPC), on an oxidized gold substrate. By monitoring the frequencies and intensities of vibrational absorption modes due to phosphate and methylene functional groups in the head and tail regions of the phospholipid, we differentiated the adsorption state of the precursor vesicles (i.e., intact vs ruptured vesicles) as a function of vesicular size in the SUV limit and the properties of the aqueous-phase solvent. We found that on oxidized gold, vesicles of DPPC in ultra pure water remained intact for all sizes tested (viz., 65, 80, and 160 nm) with varying degree of deformation. In contrast, when phosphate-buffered saline (PBS) was used as a bathing medium, all vesicles remained intact, were more distorted than the same size in pure water, and appeared to be nearly fully collapsed. Taken together, these results provide a rough guide for controlling vesicular behavior at the oxidized gold surface.

DOI: 10.1021/jp508881q

2014

Visualization of phase coexistence in the β region of cholesterol–phospholipid mixtures consisting of high cholesterol concentrations has proved elusive in lipid bilayers. Here, using the solvent-assisted lipid bilayer approach to prepare supported membranes with high cholesterol fractions close to the cholesterol solubility limit, we report the observation of coexisting liquid phases using fluorescence microscopy. At ∼63 mol % cholesterol, supported membranes consisting of mixtures of DOPC and cholesterol exhibit large-area striping reminiscent of the stripe superstructures that characterize the proximity of the second critical point in the miscibility phase diagram. The properties of the two phases are consistent with condensed complex-rich and cholesterol-rich liquids. Both phases exhibit long-range lateral mobility, and diffusion through a given phase is favored over hopping across the phase boundary, producing an “archipelago effect” and a complex percolation path

DOI: 10.1021/ja5082537

Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell–burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation. During bursting, solute leakage through transient pores relaxes the pressure and tension, replacing the domain texture by a uniform one. This isothermal phase transition—resulting from a well-coordinated sequence of mechanochemical events—suggests a complex emergent behavior allowing synthetic vesicles produced from simple components, namely, water, osmolytes, and lipids to sense and regulate their micro-environment.

DOI: 10.7554/eLife.03695.001

This paper describes the application of a solvent-exchange method to prepare supported membranes containing high fractions of cholesterol (up to ∼57 mol %) in an apparent equilibrium. The method exploits the phenomenon of reverse-phase evaporation, in which the deposition of lipids in alcohol (e.g., isopropanol) is followed by the slow removal of the organic solvent from the water-alcohol mixture. This in turn induces a series of lyotropic phase transitions successively producing inverse-micelles, monomers, micelles, and vesicles in equilibrium with supported bilayers at the contacting solid surface. By using the standard cholesterol depletion by methyl-β-cyclodextrin treatment, a quartz crystal microbalance with dissipation monitoring assay confirms that the cholesterol concentration in the supported membranes is comparable to that in the surrounding bulk phase. A quantitative characterization of the biophysical properties of the resultant bilayer, including lateral diffusion constants and phase separation, using epifluorescence microscopy and atomic force microscopy establishes the formation of laterally contiguous supported lipid bilayers, which break into a characteristic domain-pattern of coexisting phases in a cholesterol concentration-dependent manner. With increasing cholesterol fraction in the supported bilayer, the size of the domains increases, ultimately yielding two-dimensional cholesterol bilayer domains near the solubility limit. A unique feature of the approach is that it enables preparation of supported membranes containing limiting concentrations of cholesterol near the solubility limit under equilibrium conditions, which cannot be obtained using conventional techniques (i.e., vesicle fusion).

DOI: 10.1021/la5034433

We have devised an infrared spectromicroscopy based experimental configuration to enable structural characterization of buried molecular junctions. Our design utilizes a small mercury drop at the focal point of an infrared microscope to act as a mirror in studying metal-molecule-metal (MmM) junctions. An organic molecular monolayer is formed either directly on the mercury drop or on a thin, infrared (IR) semi-transparent layer of Au deposited onto an IR transparent, undoped silicon substrate. Following the formation of the monolayer, films on either metal can be examined independently using specular reflection spectroscopy. Furthermore, by bringing together the two monolayers, a buried molecular bilayer within the MmM junction can be characterized. Independent examination of each half of the junction prior to junction formation also allows probing any structural and/or conformational changes that occur as a result of forming the bilayer. Because our approach allows assembling and disassembling microscopic junctions by forming and withdrawing Hg drops onto the monolayer covered metal, spatial mapping of junctions can be performed simply by translating the location of the derivatized silicon wafer. Finally, the applicability of this technique for the longer-term studies of changes in molecular structure in the presence of electrical bias is discussed.

DOI: 10.1063/1.4896477

The entrapment of nanolipoprotein particles (NLPs) and liposomes in transparent, nanoporous silica gel derived from the precursor tetramethylorthosilicate was investigated. NLPs are discoidal patches of lipid bilayer that are belted by amphiphilic scaffold proteins and have an average thickness of 5 nm. The NLPs in this work had a diameter of roughly 15 nm and utilized membrane scaffold protein (MSP), a genetically altered variant of apolipoprotein A-I. Liposomes have previously been examined inside of silica sol-gels and have been shown to exhibit instability. This is attributed to their size (∼150 nm) and altered structure and constrained lipid dynamics upon entrapment within the nanometer-scale pores (5-50 nm) of the silica gel. By contrast, the dimensional match of NLPs with the intrinsic pore sizes of silica gel opens the possibility for their entrapment without disruption. Here we demonstrate that NLPs are more compatible with the nanometer-scale size of the porous environment by analysis of lipid phase behavior via fluorescence anisotropy and analysis of scaffold protein secondary structure via circular dichroism spectroscopy. Our results showed that the lipid phase behavior of NLPs entrapped inside of silica gel display closer resemblance to its solution behavior, more so than liposomes, and that the MSP in the NLPs maintain the high degree of α-helix secondary structure associated with functional protein-lipid interactions after entrapment. We also examined the effects of residual methanol on lipid phase behavior and the size of NLPs and found that it exerts different influences in solution and in silica gel; unlike in free solution, silica entrapment may be inhibiting NLP size increase and/or aggregation. These findings set precedence for a bioinorganic hybrid nanomaterial that could incorporate functional integral membrane proteins.

DOI: 10.1063/1.4896477

During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of ​cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding.

DOI: 10.1038/ncomms5507

Substrate-mediated fusion of small polymersomes, derived from mixtures of lipids and amphiphilic block copolymers, produces hybrid, supported planar bilayers at hydrophilic surfaces, monolayers at hydrophobic surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces, directly responding to local measures of (and variations in) surface free energy. Despite the large thickness mismatch in their hydrophobic cores, the hybrid membranes do not exhibit microscopic phase separation, reflecting irreversible adsorption and limited lateral reorganization of the polymer component. With increasing fluid-phase lipid fraction, these hybrid, supported membranes undergo a fluidity transition, producing a fully percolating fluid lipid phase beyond a critical area fraction, which matches the percolation threshold for the immobile point obstacles. This then suggests that polymer-lipid hybrid membranes might be useful models for studying obstructed diffusion, such as occurs in lipid membranes containing proteins.

DOI: 10.1021/ja5037308

The collapse of phase-separating single, supported lipid bilayers, consisting of mixtures of a zwitterionic phospholipid (POPC) and an anionic lipid (DPPA) upon thermal annealing in the presence of ions is examined using a combination of scanning probe, epifluorescence, and ellipsometric microscopies. We find that thermal annealing in the presence of ions in the bathing medium induces an irreversible transition from domain-textured, single supported bilayers to one comprising islands of multibilayer stacks, whose lateral area decays with lamellarity, producing pyramidal staircase “mesa” topography. The higher order lamellae are almost invariably localized above the anionic-lipid rich, gel-phase domains in the parent bilayer and depends on the ions in the bathing medium. The collapse mechanism appears to involve synergistic influences of two independent mechanisms: (1) stabilization of the incipient headgroup–headgroup interface in the emergent multibilayer configuration facilitated by ions in the bath and (2) domain-boundary templated folding. This collapse mechanism is consistent with previous theoretical predictions of topography-induced rippling instability in collapsing lipid monolayers and suggests the role of the mismatch in height and/or spontaneous curvature at domain boundaries in the collapse of phase-separated single supported bilayers.

DOI: 10.1021/la5005424

Borrowing principles of anhydrobiosis, we have developed a technique for self-assembling proteolipid-supported membranes on demand—simply by adding water. Intact lipid- and proteolipid vesicles dispersed in aqueous solutions of anhydrobiotic trehalose are vitrified on arbitrary substrates, producing glassy coats encapsulating biomolecules. Previous efforts establish that these carbohydrate coats arrest molecular mobilities and preserve native conformations and aggregative states of the embedded biomolecules, thereby enabling long-term storage. Subsequent rehydration, even after an extended period of time (e.g., weeks), devitrifies sugar—releasing the cargo and unmasking the substrate surface—thus triggering substrate-mediated vesicle fusion in real time, producing supported membranes. Using this method, arrays of membranes, including those functionalized with membrane proteins, can be readily produced in situ by spatially addressing vitrification using common patterning tools—useful for multiplexed or stochastic sensing and assaying of target interactions with the fluid and functional membrane surface.

DOI: 10.1021/ja410866w

Submicrometer, porous polymeric vesicles, composed of an amphiphilic di-block copolymer, polystyrene-b-polyisocyanoalanine (2-thiophene-3-yl-ethyl) amide, are used to encapsulate an enzyme, alkaline phosphatase, and a fluorescent reporter polymer poly 1(3((4methylthiophen-3-yl)oxy) propyl) quinuclidin-1-ium. Passive diffusion of exogenously added adenosine triphosphate (ATP) through the membrane was sensed by monitoring the ATP-induced fluorescence quenching of the reporter polymer followed by partial recovery of its emission due to hydrolysis of reporter-bound ATP by alkaline phosphatase.

DOI: 10.1002/smll.201300060

2013

Using lithographically defined surfaces consisting of hydrophilic patterns of nanoporous and nonporous (bulk) amorphous silica, we show that fusion of small, unilamellar lipid vesicles produces a single, contiguous, fluid bilayer phase experiencing a predetermined pattern of interfacial interactions. Although long-range lateral fluidity of the bilayer, characterized by fluorescence recovery after photobleaching, indicates a nominally single average diffusion constant, fluorescence microscopy-based measurements of temperature-dependent onset of fluidity reveals a locally enhanced fluidity for bilayer regions supported on nanoporous silica in the vicinity of the fluid–gel transition temperature. Furthermore, thermally quenching lipid bilayers composed of a binary lipid mixture below its apparent miscibility transition temperature induces qualitatively different lateral phase separation in each region of the supported bilayer: The nanoporous substrate produces large, microscopic domains (and domain-aggregates), whereas surface texture characterized by much smaller domains and devoid of any domain-aggregates appears on bulk glass-supported regions of the single-lipid bilayer. Interestingly, lateral distribution of the constituent molecules also reveals an enrichment of gel-phase lipids over nanoporous regions, presumably as a consequence of differential mobilities of constituent lipids across the topographic bulk/nanoporous boundary. Together, these results reveal that subtle local variations in constraints imposed at the bilayer interface, such as by spatial variations in roughness and substrate adhesion, can give rise to significant differences in macroscale biophysical properties of phospholipid bilayers even within a single, contiguous phase.

DOI: 10.1021/ja408434r

We have studied the interaction of the enzyme sphingomyelinase with sphingomyelin-containing supported membranes using quantitative applications of real-time epifluorescence microscopy and imaging optical ellipsometry. The enzymatic action converts sphingomyelin into ceramides by cleaving the phosphodiester bond. Our results confirm previous studies establishing a gross morphological transformation of lipid bilayers involving a multi-step process consisting of lag-burst type of enzyme activation and in-plane reorganization of membrane components attributed to the formation of ceramide-enriched domains. A unique finding of our study is the evidence for the existence of an additional out-of-plane deformation following lateral reorganization resulting in membrane voids disrupting the laterally contiguous bilayer. Taken together, the in-plane and out-of-plane deformations suggest a mechanistic picture in which lateral diffusional processes of translational mobility and phase separation couple with out-of-plane interactions across the membrane leaflet to induce irreversible membrane disruption in response to SMase action. Remarkably, lipid monolayers supported on hydrophobic substrates exhibit no such large-scale deformation despite ceramide generation by enzymatic activity of sphingomyelinase, possibly suggesting the importance of coupling across membrane leaflets in inducing out-of-plane deformations.

DOI: 10.1039/C3SM51855H

Hybrid phospholipid/block copolymer vesicles, in which the polymeric membrane is blended with phospholipids, display interesting self-assembly behavior, incorporating the robustness and chemical versatility of polymersomes with the softness and biocompatibility of liposomes. Such structures can be conveniently characterized by preparing giant unilamellar vesicles (GUVs) via electroformation. Here, we are interested in exploring the self-assembly and properties of the analogous nanoscale hybrid vesicles (ca. 100 nm in diameter) of the same composition prepared by film-hydration and extrusion. We show that the self-assembly and content-release behavior of nanoscale polybutadiene-b-poly(ethylene oxide) (PB-PEO)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) hybrid phospholipid/block copolymer vesicles can be tuned by the mixing ratio of the amphiphiles. In brief, these hybrids may provide alternative tools for drug delivery purposes and molecular imaging/sensing applications and clearly open up new avenues for further investigation.

DOI: 10.1039/C3SM51855H

This Letter describes Fourier-transform infrared spectroscopy evidence for the evolution of conformational order and coverage during the formation of n-alkanethiol monolayers on microdroplets of mercury from the solution phase. At the highest coverages obtained by self-assembly, the monolayer is characterized by predominantly all-trans conformational order. For partial monolayers obtained at arbitrarily quenched incubation periods, we find a continuous evolution of the chain conformational order with monolayer coverage. Analyzing these results in light of previously reported models from X-ray scattering reveals a complex self-assembly process in which the density-dependent evolution of the chain conformational order is coupled with that of molecular orientation and density.

DOI: 10.1021/la4014366

We have studied interfacial compressibility and lateral organization in monolayer configurations of total (squalene containing) and polar (squalene-devoid) lipid extracts of Halobacterium salinarum NRC-1, an extremely halophilic archaeon. Pressure–area isotherms derived from Langmuir experiments reveal that packing characteristics and elastic compressibility are strongly influenced by the presence of squalene in the total lipid extract. In conjunction with control experiments using mixtures of DPhPC and squalene, our results establish that the presence of squalene significantly extends elastic area compressibility of total lipid extracts, suggesting it has a role in facilitating tighter packing of archaeal lipid mixtures. Moreover, we find that squalene also influences spatial organization in archaeal membranes. Epifluorescence and atomic force microscopy characterization of Langmuir monolayers transferred onto solid hydrophilic substrates reveal an unusual domain morphology. Individual domains of microscopic dimensions (as well as their extended networks) exhibiting a peculiar bowl-like topography are evident in atomic force microscopy images. The tall rims outlining individual domains indicate that squalene accumulates at the domain periphery in a manner similar to the accumulation of cholesterol at domain boundaries in their mixtures with phospholipids. Taken together, the results presented here support the notion that squalene plays a role in modulating molecular packing and lateral organization (i.e., domain formation) in the membranes of archaea analogous to that of cholesterol in eukaryotic membranes.

DOI: 10.1021/la401412t

Conjugated polyelectrolytes (CPEs) are promising materials for generating optoelectronics devices under environmentally friendly processing conditions, but challenges remain to develop methods to define lateral features for improved junction interfaces and direct optoelectronic pathways. We describe here the potential to use a bottom-up approach that employs self-assembly in lipid membranes to form structures to template the selective adsorption of CPEs. Phase separation of gel phase anionic lipids and fluid phase phosphocholine lipids allowed the formation of negatively charged domain assemblies that selectively adsorb a cationic conjugated polyelectrolyte (P2). Spectroscopic studies found the adsorption of P2 to negatively charged membranes resulted in minimal structural change of the solution phase polymer but yielded an enhancement in fluorescence intensity (∼50%) due to loss of quenching pathways. Fluorescence microscopy, dynamic light scattering, and AFM imaging were used to characterize the polymer–membrane interaction and the polymer-bound domain structures of the biphasic membranes. In addition to randomly formed circular gel phase domains, we also show that predefined features, such as straight lines, can be directed to form upon etched patterns on the substrate, thus providing potential routes toward the self-organization of optoelectronic architectures.

DOI: 10.1021/la400454c

We report the experimental observation of osmotically induced transient pearling instabilities in vesicular membranes. Giant phospholipid vesicles subjected to negative osmotic gradient, which drives the influx of water in to the vesicular interior, produces transient cylindrical protrusions. These protrusions exhibit a remarkable pearling intermediate, which facilitates their subsequent retraction. The pearling front propagates from the distal free end of the protrusion toward the vesicular source and accompanies gradual shortening of the protrusion via pearl–pearl coalescence. Real-time introduction of a positive osmotic gradient, on the other hand, drives vigorous shape fluctuations, which in turn produce cylindrical, prolate- and pear-shaped intermediates presumably due to an increased vesicular area relative to the encapsulated volume. These intermediates transiently produce a pearled state prior to their fission. In both cases, the transient pearling state gives rise to an array of stable spherical daughter vesicles, which may be connected to one another by fine tethers not resolved in our experiments. These results may have implications for self-reproduction in primitive, protein-free, cells.

DOI: 10.1039/C2FD20116J

A recent experimental study [1] has demonstrated the alignment of phase separated domains across hundreds of bilayer units in multicomponent stacked lipid bilayers. The origin of this alignment is the interlamellar coupling of laterally phase separated domains. Here, we develop a theoretical model that presents the energetics description of this phenomenon based on the minimization of the free energy of the system. Specifically, we use solution theory to estimate the competition between energy and entropy in different stacking configurations. The model furnishes an elemental phase diagram, which maps the domain distributions in terms of the strength of the intra- and inter-layer interactions and estimates the value of inter-layer coupling for complete alignment of domains in the stacks of five and ten bilayers. The area fraction occupied by co-existing phases was calculated for the system of the minimum free energy, which showed a good agreement with experimental observations.

DOI: 10.3390/ijms14023824

2012

Liquid-crystalline phases of stacked lipid bilayers represent a pervasive motif in biomolecular assemblies. Here we report that, in addition to the usual smectic order, multicomponent multilayer membranes can exhibit columnar order arising from the coupling of two-dimensional intralayer phase separation and interlayer smectic ordering. This coupling propagates across hundreds of membrane lamellae, producing long-range alignment of phase-separated domains. Quantitative analysis of real-time dynamical experiments reveals that there is an interplay between intralayer domain growth and interlayer coupling, suggesting the existence of cooperative multilayer epitaxy. We postulate that such long-range epitaxy is solvent-assisted, and that it originates from the surface tension associated with differences in the network of hydrogen-bonded water molecules at the hydrated interfaces between the domains and the surrounding phase. Our findings might inspire the development of self-assembly-based strategies for the long-range alignment of functional lipid domains.

DOI: 10.1038/nmat3451

Molecular-level control over surface chemistry and topology is critical for the design of biologically active synthetic surfaces. Such surfaces must present active biological ligands in defined conformations, orientations, concentrations, and spatial distributions so as to foster biospecific interactions and inhibit nonspecific ones. Self-assembled monolayers (SAMs)-spontaneously organized monomolecular assemblies at solid surfaces-provide an elegant and versatile means to endow synthetic surfaces with such exquisite level of control at the molecular level. This chapter reviews the essential physical-chemical foundation for the preparation, structure, and formation mechanisms of SAMs; presents their amenability for spatial control using tools of micro-and nanopatterning; and highlights their enabling capacity for a broad range of biomolecular functionalization.

DOI: 10.4032/9789814364188

Using epifluorescence microscopy, the configuration of myelin figures that are formed upon hydration of lipid stack was studied qualitatively. Little knowledge is currently available for conditions that determine the diameter of myelin figures and their degree of multilamellarity. Examining more than 300 samples, we realized that there are distinct populations of myelin figures protruding from discrete regions of lipid stack. Each population contains myelin figures with similar diameters. This indicates a direct relationship between local characteristics of parent lipid stack and the diameter of myelin figures. Evidenced by fluorescent images, we classified all the observed myelin figures into three major groups of (1) solid tubes, (2) thin tethers, and (3) hollow tubes. Solid tubes are the most common structure of myelin figures which appeared as dense shiny cylinders. Thin tethers, with long hair-shaped structure, were observed protruding from part of lipid plaque which is likely to be under tension. Hollow tubes were protruded from the parts that are unpinned from the substrate and possibly under low or no tension. The abrupt change in the configuration of myelin figures from solid tubes to hollow ones was described in a reproducible experiment where the pinned region of the parent stack became unpinned. Our observations can indicate a relation between the membrane tension of the source material and the diameter of the myelin figures.

DOI: 10.4032/9789814364188

Background| The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer's disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species. Methodology/Principal Findings| To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species. Conclusions| Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.

DOI: 10.1371/journal.pone.0035443

We report observations of large-scale, in-plane and out-of-plane membrane deformations in giant uni- and multilamellar vesicles composed of binary and ternary lipid mixtures in the presence of net transvesicular osmotic gradients. The lipid mixtures we examined consisted of binary mixtures of DOPC and DPPC lipids and ternary mixtures comprising POPC, sphingomyelin and cholesterol over a range of compositions – both of which produce co-existing phases for selected ranges of compositions at room temperature under thermodynamic equilibrium. In the presence of net osmotic gradients, we find that the in-plane phase separation potential of these mixtures is non-trivially altered and a variety of out-of-plane morphological remodeling events occur. The repertoire of membrane deformations we observe display striking resemblance to their biological counterparts in live cells encompassing vesiculation, membrane fission and fusion, tubulation and pearling, as well as expulsion of entrapped vesicles from multicompartmental giant unilamellar vesicles through large, self-healing transient pores. These observations suggest that the forces introduced by simple osmotic gradients across membrane boundaries could act as a trigger for shape-dependent membrane and vesicle trafficking activities. We speculate that such coupling of osmotic gradients with membrane properties might have provided lipid-mediated mechanisms to compensate for osmotic stress during the early evolution of membrane compartmentalization in the absence of osmoregulatory protein machinery.

DOI: 10.3389/fphys.2012.00120

Native vesicles or “reduced protocells” derived by mechanical extrusion concentrate selected plasma membrane components, while downsizing complexities of whole cells. We illustrate this technique, characterize the physical-chemical properties of these reduced configurations of whole cells, and demonstrate their surface immobilization and patternability. This simple detergent-free vesicularized membrane preparation should prove useful in fundamental studies of cellular membranes, and may provide a means to engineer therapeutic cells and enable high-throughput devices containing near-native, functional proteolipidic assemblies.

DOI: 10.1039/C2IB20022H

Attenuated total reflectance Fourier transform infrared spectroscopy is used to monitor the adsorption of 100 nm 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipid vesicles to the surfaces of Ge, electrolessly deposited Au, and a well formed self-assembled monolayer of 1-octadecanethiol. The interaction of DPPC vesicles in solution with these different surfaces yields distinctly different surface structures: intact DPPC vesicles on Ge, a supported phospholipid bilayer on an electrolessly deposited Au surface, and a phospholipid monolayer onto the hydrophobic self-assembled monolayer. IR peak position, bandwidth, and intensity are used to confirm structure formation and quantitation of the amount of lipid that desorbs during film formation.

DOI: 10.1364/AO.51.002842

Using spatially patterned supported lipid mono- and bilayers, we compare the effect of transleaflet dynamics on membrane solubilization by a common, non-ionic detergent in single samples. We find that at concentrations surrounding CMC, complete bilayers undergo 5–8% lateral expansion followed by rapid dissolution. In contrast, single supported monolayers remain remarkably resistant to solubilization, suggesting the central role of detergent or lipid flip-flop in driving membrane solubilization. In addition to the previously well-appreciated mode of detergent-resistance by tight lateral packing of saturated and cholesterol-rich lipids (e.g., rafts) in membrane bilayers, our results suggest that hindrance to interleaflet dynamics, such as by strong interaction with the cytoskeleton, provides an alternative mechanism by which membranes resist detergent solubilisation. Furthermore, we show that this differential resistance can be exploited to design spatial compositional patterns of lipid bilayers and monolayers.

DOI: 10.1039/C2SM00025C

Attenuated total reflection Fourier transform infrared spectroscopy was used to monitor the formation of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), DMPC: lactosylceramide, and DMPC: GD3 lipid bilayers onto a zinc selenide surface. Infrared absorption peak position, bandwidth, and intensity were all used to monitor the formation, acyl chain ordering, and chemical environment within each bilayer. The results from this study indicate that the addition of glycosphingolipids into a DMPC lipid bilayer introduces decreases in both, acyl chain ordering, and homogeneity within the bilayer. GD3:DMPC lipid bilayers possess lipid chain characteristics that are indiscernible from those present in the lactosylceramide:DMPC bilayer, while possessing different structural head groups, indicating that the head group has little influence on the underlying lipid structure. Differences in the phosphate hydration are, however, evident between the three types of bilayer, with phosphate hydration decreasing in the order LacCer:DMPC (1223.4 cm(-1)) > DMPC only (1226 cm(-1)) > GD3:DMPC (1229.6 cm(-1))

DOI: 10.1016/j.colsurfb.2012.01.034

Virulent strains of bacteria and viruses recognize host cells by their plasma membrane receptors and often exploit the native translocation machinery to invade the cell. A promising therapeutic concept for early interruption of pathogen infection is to subvert this pathogenic trickery using exogenously introduced decoys that present high-affinity mimics of cellular receptors. This review highlights emerging applications of molecularly engineered lipid-bilayer-based nanostructures, namely (i) functionalized liposomes, (ii) supported colloidal bilayers or protocells and (iii) reconstituted lipoproteins, which display functional cellular receptors in optimized conformational and aggregative states. These decoys outcompete host cell receptors by preferentially binding to and neutralizing virulence factors of both bacteria and viruses, thereby promising a new approach to antipathogenic therapy.

DOI: 10.1016/j.tibtech.2012.03.002

Lipid molecules in water form uni- or multilamellar vesicles in polydisperse form. Herein, we present energetic considerations for their equilibrium morphological organization. Our formulation provides elemental energy diagrams, which explain the polydispersity and account for the structural diversity. These energy diagrams describe the ranges of core radius (rc) and number of lamellae (N) that result in the formation of stable vesicles under specific conditions, thus providing prescriptions for the design of vesicles tailored for specific properties, including stability, cargo capacity, and resistance to deformation by osmotic stress. We deduced key design criteria as follows: 1) designing highly stable unilamellar vesicles requires low bending rigidity lipids and dimensions exceeding a few hundred nm in radii; 2) very large unilamellar vesicles (rc>several tens of microns) are not stable for typical lipids; lipids with higher bending rigidity are required; 3) the distribution of the stable size of vesicles is proportional to the bending rigidity; 4) for the case of multilamellar vesicles, vesicles with more than a few hundred layers usually exhibit greater structural integrity than those with lower degrees of lamellarity, especially when the core radii are small (less than 100 nm); 5) for osmotically stressed vesicles, the energy contributed by even a small concentration gradient (>mM) is the most dominant factor in the free energy, suggesting active response by vesicles (e.g., poration) to release osmotic stress; and 6) vesicles with a core radius of a few hundred nm and more than hundred lamellae are more resistant to deformation by osmotic stress, thus making them more suited to applications involving osmotic pressure gradients, such as in drug delivery.

DOI: 10.1002/cphc.201100573

2011

In living cells, mechanochemical coupling represents a dynamic means by which membrane components are spatially organized. An extra-ordinary example of such coupling involves curvature-dependent polar localization of chemically-distinct lipid domains at bacterial poles, which also undergo dramatic reequilibration upon subtle changes in their interfacial environment such as during sporulation. Here, we demonstrate that such interfacially-triggered mechanochemical coupling can be recapitulated in vitro by simultaneous, real-time introduction of mechanically-generated periodic curvatures and attendant strain-induced lateral forces in lipid bilayers supported on elastomeric substrates. In particular, we show that real-time wrinkling of the elastomeric substrate prompts a dynamic domain reorganization within the adhering bilayer, producing large, oriented liquid-ordered domains in regions of low curvature. Our results suggest a mechanism in which interfacial forces generated during surface wrinkling and the topographical deformation of the bilayer combine to facilitate dynamic reequilibration prompting the observed domain reorganization. We anticipate this curvature-generating model system will prove to be a simple and versatile tool for a broad range of studies of curvature-dependent dynamic reorganizations in membranes that are constrained by the interfacial elastic and dynamic frameworks such as the cell wall, glycocalyx, and cytoskeleton.

DOI: 10.1371/journal.pone.0028517

We have fabricated a stack of five 1,2-dipalmitoyl-sn-3-phosphatidylethanolamine (DPPE) bilayers supported on a polished silicon substrate in excess water. The density profile of these stacks normal to the substrate was obtained through analysis of x-ray reflectivity. Near the substrate, we find the layer roughness and repeat spacing are both significantly smaller than values found in bulk multilayer systems. The reduced spacing and roughness result from suppression of lateral fluctuations due to the flat substrate boundary. The layer spacing decrease then occurs due to reduced Helfrich repulsion.

DOI: 10.1103/PhysRevE.84.041914

A robust and straightforward method for the preparation of lipid membranes upon dynamically responsive polymer cushions is reported. Structural characterization demonstrates that complete, well-packed membranes with tunable mobility can be constructed on the polymeric cushion. With this system, membrane conformational changes induced by cellular cytoskeleton interactions can be modeled. The membrane can be tailored to screen the cushion from changes in pH or allow rapid response to the pH environment by incorporation of protein ion channels. This elementary system offers a means to replicate the conformational changes that occur with the cellular cytoskeleton and has great potential for fundamental biophysical studies of membrane properties and membrane-protein interactions decoupled from the underlying solid support.

DOI: 10.1021/n1200832c

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes.

DOI: 10.1038/nmat2992

We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics.

DOI: 10.1073/pnas.1009043108

One of biology’s most pervasive nanostructures, the phospholipid membrane, represents an ideal scaffold for a host of nanotechnology applications. Whether engineering biomimetic technologies or designing therapies to interface with the cell, this adaptable membrane can provide the necessary molecular-level control of membrane-anchored proteins, glycopeptides, and glycolipids. If appropriately prepared, these components can replicate in vitro or influence in vivo essential living processes such as signal transduction, mass transport, and chemical or energy conversion. To satisfy these requirements, a lipid-based, synthetic nanoscale architecture with molecular-level tunability is needed. In this regard, discrete lipid particles, including reconstituted high density lipoprotein (HDL), have emerged as a versatile and elegant solution. Structurally diverse, native biological HDLs exist as discoidal lipid bilayers of 5−8 nm diameter and lipid monolayer-coated spheres 10−15 nm in diameter, all belted by a robust scaffolding protein. These supramolecular assemblies can be reconstituted using simple self-assembly methods to incorporate a broad range of amphipathic molecular constituents, natural or artificial, and provide a generic platform for stabilization and transport of amphipathic and hydrophobic elements capable of docking with targets at biological or inorganic surfaces. In conjunction with top-down or bottom-up engineering approaches, synthetic HDL can be designed, arrayed, and manipulated for a host of applications including biochemical analyses and fundamental studies of molecular structure. Also highly biocompatible, these assemblies are suitable for medical diagnostics and therapeutics. The collection of efforts reviewed here focuses on laboratory methods by which synthetic HDLs are produced, the advantages conferred by their nanoscopic dimension, and current and emerging applications.

DOI: 10.1021/nn103098m

We report the observation of an unusual stripe−droplet transition in precompressed Langmuir monolayers consisting of mixtures of poly(ethylene) glycol (PEG) amphiphiles and phospholipids. This highly reproducible and fully reversible transition occurs at approximately zero surface pressure during expansion (or compression) of the monolayer following initial compression into a two-dimensional solid phase. It is characterized by spontaneous emergence of an extended, disordered stripe-like morphology from an optically homogeneous phase during gradual expansion. These stripe patterns appear as a transient feature and continuously progress, involving gradual coarsening and ultimate transformation into a droplet morphology upon further expansion. Furthermore, varying relative concentrations of the two amphiphiles and utilizing amphiphiles with considerably longer ethylene glycol headgroups reveal that this pattern evolution occurs in narrow concentration regimes, values of which depend on ethylene oxide headgroup size. These morphological transitions are reminiscent of those seen during a passage through a critical point by variations in thermodynamic parameters (e.g., temperature or pressure) as well as those involving spinodal decomposition. While the precise mechanism cannot be ascertained using present experiments alone, our observations can be reconciled in terms of modulations in competing interactions prompted by the pancake−mushroom−brush conformational transitions of the ethylene glycol headgroup. This in turn suggests that the conformational degree of freedom represents an independent order parameter, or a switch, which can induce large-scale structural reorganization in amphiphilic monolayers. Because molecular conformational changes are pervasive in biological membranes, we speculate that such conformational transition-induced pattern evolution might provide a physical mechanism by which membrane processes are amplified.

DOI: 10.1021/la104175f

We report a hybrid drug delivery system inspired by the functional compartmentalization of cell, isolating properties of cargo encapsulation, targeting, stability, biocompatibility, and permeability into discrete multilamellar organic-inorganic–organic design consisting of two differently functionalized lipid bilayers sandwiching a nanoporous silica layer.

DOI: 10.1002/cphc.201100573

2010

The authors have studied microstructure evolution during thermally induced phase separation in a class of binary supported lipid bilayers using a quantitative application of imaging ellipsometry. The bilayers consist of binary mixtures consisting of a higher melting glycosphingolipid, galactosylceramide (GalCer), which resides primarily in the outer leaflet, and a lower melting, unsaturated phospholipid, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). Three different bilayer compositions of GalCer/DLPC mixtures at 35:65, 20:80, and 10:90 molar ratios were cooled at controlled rates from their high-temperature homogeneous phase to temperatures corresponding to their phase coexistence regime and imaged in real time using imaging ellipsometry. During the thermotropic course of GalCer gelation, we find that two distinct types of morphological features modulate. First, the formation and growth of chain and fractal-like defects ascribed to the net change in molecular areas during the phase transition. The formation of these defects is consistent with the expected contraction in the molecular area during the liquid crystalline to gel-phase transition. Second, the nucleation and growth of irregularly shaped gel-phase domains, which exhibit either line-tension dominated compact shape or dendritic domains with extended interfaces. Quantifying domain morphology within the fractal framework reveals a close correspondence, and the quantization of the transition width confirms previous estimates of reduced phase transition cooperativity in supported bilayers. A comparison of domain properties indicates that thermal history, bilayer composition, and cooling rate all influence microstructure details including shapes, sizes, and distributions of domains and defects: At lower cooling rates and lower GalCer fractions compact domains form and at higher GalCer fractions (or at higher cooling rates) dendritic domains are evident. This transition of domain morphology from compact shapes to dendritic shapes at higher cooling rates and higher relative fractions of GalCer suggests kinetic control of shape equilibration in these phospho- and glycolipid mixtures.

DOI: 10.1116/1.3524295

Milk fat globules (MFGs) are accepted primarily as triacylglycerol delivery systems. The identification of nanometer-sized lipid−protein particles termed “lactosomes” that do not contain triacylglycerol raises the question of their possible functions. MFGs were isolated by slow centrifugation, and lactosomes were isolated by ultracentrifugation at a density equivalent to plasma high-density lipoproteins (HDL) (d > 1.063 g/mL) from human milk obtained from six volunteers at different lactation stages. Isolated lactosomes were analyzed and compared with MFGs for their size distribution, lipidome, proteome, and functional activity. Lactosomes from early milk, day 8, were found to be similar in size as those from mature milk >28 days, averaging ∼25 nm in diameter. In total, 97 nonredundant proteins were identified in the MFG and lactosome fractions, 46 of which were unique to the MFG fraction and 29 of which were unique to the lactosome fraction. The proteins identified in the lactosome and MFG fractions were enriched with proteins identified with immunomodulatory pathways. Unlike MFGs and GM1-laden reconstituted HDL that served as a positive control, lactosomal binding capacity to cholera toxin was weak. Lipidomic analyses found that lactosomes were devoid of triacylglycerol and gangliosides, unlike MFGs, but rich in a variety of phospholipid species. The data found differences in structure, composition, and function between lactosomes and MFG, suggesting that these two particles are derived from different biosynthetic and/or secretory pathways. The results reveal a bioactive lipid−protein, nanometer-length scale particle that is secreted into milk not to supply energy to the infant but to play unique, protective, and regulatory roles.

DOI: 10.1021/jf102495s

Lipid monolayers and bilayers exist in distinct physical states differentiated by the differences in the manner in which translational fluidity relates to their phase transition and how cholesterol influences the two. Work presented here suggests that intra-leaflet diffusion and cholesterol interactions are modulated by the nature of inter-leaflet coupling. Our results also provide an important practical caveat in the comparisons of membrane physical properties deduced using the two, mono- and bilayer, model membrane configurations.

DOI:10.1039/C0SM00643B

Phospholipid membranes assembled on a planar colloidal crystal functionalized with a proton sensitive fluorescence probe offers a useful model system for a convenient, on-chip, parallel, and fluorescence based assay of membrane-mediated ion transport. The fusion of small unilamellar vesicles on surface derivatized hydrophilic/hydrophobic planar colloidal crystals results in a single lipid bilayer on the hydrophilic region and a monolayer on the hydrophobic region. We show that this composite structure exhibits a laterally continuous outer leaflet spanning the two mono- and bilayer morphologies, thereby providing an effective seal against ionic permeability. This is in contrast to the structure of lipids assembled on a patterned wettability planar coverslip where there a lipophobic “moat” formed at the junction between the mono- and the bilayer configurations prevents forming well-sealed membrane. By embedding a pH sensitive fluorescent probe, namely, fluorescein, within the colloidal crystal interstices, we find that the colloidal crystal supported membrane configuration provides a practical assay for optical characterization of membrane mediated ionic transport. We demonstrate the ability of our platform for the case of passive transport of protons across a fluid phospholipid bilayer. The membrane permeability coefficients derived, including those associated with the biphasic permeation mechanism, match well with those reported for vesicle based assays and confirm the quantitative optical transduction. We also illustrate the use of this experimental platform for parallel measurements such as may be useful for the characterization of stochastic transport and high throughput measurements

DOI:10.1039/C0SM00535E

We present experimental evidence for the existence of a unique molecular-level order in the vicinity of the bilayer’s edge. Discrete patches of substrate-supported lipid bilayers exhibiting stable edge defects are prepared by confining vesicle fusion to hydrophilic patches of a chemically patterned substrate exhibiting hydrophilic patches in hydrophobic surrounding, and edge properties are characterized by fluorescence and vibrational spectroscopy based measurements. Specifically, wide-field fluorescence microscopy using phase-sensitive dyes, temperature-programmed fluorescence recovery measurements, and temperature-dependent attenuated total reflection Fourier transform infrared spectroscopy measurements are performed to characterize the local chain conformational properties, local diffusional characteristics, and phase discrimination afforded by phase-sensitive DiI fluorescent probes. We find that the bilayer structure near the edge is characterized by (1) an increase in intramolecular conformational order; (2) reduced effective lateral mobility; and (3) a distinctly higher local, effective gel-fluid transition temperature in comparison to their bulk counterpart. Together, these features signal the emergence of unique ordering presumably triggered by the hemimicellar configuration of the edge. These results are consistent with simulations of lyso-lipid micelles predicting the presence of dynamic clusters of ordered lipids in comparable micellar topology and disagrees with some recent interpretations of mobility near the edges of supported bilayers. Our results also offer the structural basis for the stability of defects and edges in fluid supported bilayers, and may be relevant in understanding the ordering and stabilization of pores, edges, and defects generated in membrane bilayers by proteins, curvature-sensitive lipids, antimicrobial peptides, and detergents.

DOI:10.1021/ja100294k

The ability to exogenously present cell-surface receptors in high-affinity conformations in a synthetic system offers an opportunity to provide host cells with protection from pathogenic toxins. This strategy requires improvement of the synthetic receptor binding affinity against its native counterpart, particularly with polyvalent toxins where clustering of membrane receptors can hinder binding. Here we demonstrate that reconstituted lipoprotein, nanometer-sized discoidal lipid bilayers bounded by apolipoprotein and functionalized by incorporation of pathogen receptors, provides a means to enhance toxin-receptor binding through molecular-level control over the receptor microenvironment (specifically, its rigidity, composition, and heterogeneity). Using a Foerster Resonance Energy Transfer (FRET)-based assay, we found that reconstituted lipoprotein incorporating low concentrations of ganglioside monosialotetrahexosylganglioside (GM1) binds polymeric cholera toxin with significantly higher affinity than liposomes or supported lipid bilayers, most likely a result of the enhanced control over receptor clustering provided by the lipoprotein platform. Using wide-area epifluorescence, we found that this enhanced binding capacity can be effectively utilized to divert cholera toxin away from populations of healthy mammalian cells. In summary, we found that reconstitutions of high-density lipoprotein can be engineered to include specific pathogen receptors; that their pathogen binding affinity is altered, presumably due to attenuation of receptor aggregation; and that these assemblies are effective at protecting cells from biological toxins.

DOI:10.1194/jlr.M007401

The sol−gel processes of 3-glycidoxypropyltrimethoxysilane (GPTMS) and methacryloxypropyltrimethoxysilane (MAPTMS) have been followed by fluorescence spectroscopy with pyranine as a photophysical probe. The experimental results showed that this probe is sensitive to the structural evolution and microenvironment polarity. The specific comparison of the structural evolution in two substituted organotrialkoxysilanes, namely, MAPTMS and GPTMS, illustrates the ability of the substituents to interact with the microenvironment via electrostatic interactions. Interestingly, these interactions determine the kinds of intermediate supramolecular structures that form during the sol−gel process and hence control the structure of the ensuing sol−gel end product. In particular, the amphiphile-like character of the MAPTMS intermediates contrasts with the biamphiphilic character of their GPTMS counterparts, driving distinctly different transient and local molecular organizations, which in turn modulate the hydrolysis and condensation reactions during the sol−gel process.

DOI:10.1021/la904040c

We have investigated the response of solid-supported phospholipid bilayers to short doses of photogenerated oxidative stress to characterize physical membrane changes during early phases of membrane oxidation. The low-dose oxidative stress is generated by uniformly exposing the bilayer samples using short-wavelength UV radiation (184−257 nm) for short periods (∼3 min) and resulting membrane morphological transformations characterized using a combination of wide-field epifluorescence microscopy and imaging ellipsometry measurements. Our results establish that the early phase of membrane oxidation is characterized by the nucleation and growth of discrete microscopic voids within the bilayer. The locations of the voids are randomly distributed throughout the sample surface, despite the uniform illumination. Over longer time scales, the voids continue to grow after the termination of the UV radiation. We also find that the voids heal as sample temperature is raised and that the supported bilayers consisting of fully saturated lipids are less susceptible to the mild oxidation conditions used, regardless of phase state. Analyzing these results in terms of (1) reactive-oxygen species mediated oxidative attack, (2) in situ generation of membrane oxidation products, and (3) their reequilibration between the membrane and the bulk aqueous phase explains the membrane morphological changes observed and provides insights into membrane perturbations following oxidative assault. Specifically, molecular properties of oxidation products (e.g., intrinsic curvature) account for formation and stabilization of voids within contiguous bilayers, and the long-term structural evolution is consistent with slow kinetics of the desorption of these oxidation products from the bilayer into bulk solution. A corollary benefit from our study is that the thermal properties of voids appear to offer a useful means to measure the thermal expansivity of supported membranes.

DOI:10.1021/jp102861v

We show that a coordinated interplay between mesoscale colloidal interactions and molecular-scale membrane perturbations affords a novel material platform to controllably recapitulate membrane interactions, such as during fusion and vesicle-based drug delivery. Specifically, a simple modulation of ionic strength is used to alter electrostatically determined colloidal interactions, producing conditions for pre-fusion contact between two independent colloidal and/or planar supported lipid bilayers. The same process also perturbs the membrane at the molecular level, reproducing conditions needed for subsequent fusion steps. We envisage that this platform will yield insights relevant to optimal design and implementation of lipid-coated inorganic constructs used for therapeutic drug delivery and sensing.

DOI:10.1039/C001586E

The physical and chemical properties of biological membranes are intimately linked to their bounding aqueous interfaces. Supported phospholipid bilayers, obtained by surface-assisted rupture, fusion, and spreading of vesicular microphases, offer a unique opportunity, because engineering the substrate allows manipulation of one of the two bilayer interfaces as well. Here, we review a collection of recent efforts, which illustrates deliberate substrate–membrane coupling using structured surfaces exhibiting chemical and topographic patterns. Vesicle fusion on chemically patterned substrates results in co-existing lipid phases, which reflect the underlying pattern of surface energy and wettability. These co-existing bilayer/monolayer morphologies are useful both for fundamental biophysical studies (e.g., studies of membrane asymmetry) as well as for applied work, such as synthesizing large-scale arrays of bilayers or living cells. The use of patterned, static surfaces provides new models to design complex membrane topographies and curvatures. Dynamic switchable-topography surfaces and sacrificial trehalose based-substrates reveal abilities to dynamically introduce membrane curvature and change the nature of the membrane–substrate interface. Taken together, these studies illustrate the importance of controlling interfaces in devising model membrane platforms for fundamental biophysical studies and bioanalytical devices.

DOI:10.1016/j.bbamem.2009.12.029

In free bilayers, the fluid to gel main phase transition of a monofluorinated phospholipid (F-DPPC) transforms a disordered fluid bilayer into a fully interdigitated monolayer consisting of ordered acyl tails. This transformation results in an increase in molecular area and decrease in bilayer thickness. We show that when confined in patches near a solid surface this reorganization proceeds under constraints of planar topography and total surface area. One consequence of these constraints is to limit the complete formation of the energetically favored, interdigitated gel phase. The noninterdigitated lipids experience enhanced lateral tension, due to the expansion of the growing interdigitated phase within the constant area. The corresponding rise in equilibrium transition temperatures produces supercooled lipids that vitrify when cooled further. Ultimately, this frustrated phase change reflects a coupling between dynamics and thermodynamics and gives rise to an unusual phase coexistence characterized by the presence of two qualitatively different gel phases.

< DOI:10.1021/jp908585u

2009

IMembrane dynamics: Slip—the relative mobility of the two leaflets in lipid bilayers—represents a generic mechanical-dynamical process. To establish the presence of interleaflet slip in the surface-spreading lipid membranes, the advancing lipid bilayers are exposed to an intense laser spot, which rapidly bleaches the illuminated fluorophores in a radially symmetric shape (see figure).

DOI:10.1002/cphc.200900372

We report acceleration in the rate of bulk phase gelation of an organoalkoxysilane, 3-methacryloxypropyltrimethoxysilane (MAPTMS), in the presence of an amphiphilic additive, N-phenyl glycine (NPG). The MAPTMS gelation occurs within 30 min in the presence of 0.5 wt % NPG, which took several months in the absence of NPG. Using a combination of ATR-FT IR, (29)Si NMR, (1)H NMR, viscosity analysis, SEM, UV-vis, and pi-A isotherm measurements, we elucidate the molecular-level details of the structural changes during NPG-catalyzed MPTMS gelation rate. On the basis of these results, we propose a gelation mechanism in which a transient cooperative self-assembly process fosters hydrolysis and retards early condensation thereby promoting the formation of extended three-dimensionally cross-linked gels. Specifically, the amphiphilic character of the hydrolysis product of MAPTMS, consisting of a hydrophobic tail R = -CH(2)CH(2)CH(2)O(CO)C(CH(3)) horizontal lineCH(2) and a hydrophilic Si-OH headgroup, promotes micelle formation at high MAPTMS/water ratio. NPG readily inserts within these micelles thus retarding the topotactic condensation of silanols at the micellar surface. This in turn allows for a more complete hydrolysis of Si-OCH(3) groups prior to condensation in MAPTMS. With increased silanol concentration at the micellar periphery, a delayed condensation phase initiates. This formation of a covalently bonded Si-O-Si framework (and possibly also the formation of the methanol byproduct) likely destabilizes the micellar motif thus promoting its transformation into condensed mesophases (e.g., lamellar microstructure) upon gelation. Because of the generality of this transient and co-operative organic-inorganic self-assembly between hydrolyzed amphiphilic organoalkoxysilanes and surfactant-like amino acid additives, we envisage applications in controlling bulk phase gelation of many chain-substituted organoalkoxysilanes.

DOI:10.1021/jp905069j

The natural cell cytoskeleton provides critical mechanical support for lipid bilayers using a network of actin fibers. As a step towards fabricating artificial cells, functional nanofiber networks were created to mimic the chemical and mechanical environment provided by the actin network. Lipid bilayers were formed on a random nanofiber mat made from [similar]131 nm diameter polycaprolactam nanofibers modified to be highly hydrophilic by coating with 6.6 nm of silica (SiO2). The nanofiber-supported bilayers were characterized using fluorescence recovery after photobleaching (FRAP) and electrophysiological recordings and found to be comparable to bilayer recordings from natural membranes. The nanofiber-supported bilayers were also more stable than conventional, unsupported, black lipid bilayers.

DOI:10.1039/B903048D

This paper describes a novel surface engineering approach that combines oxygen plasma treatment and electrochemical activation to create micropatterned cocultures on indium tin oxide (ITO) substrates. In this approach, photoresist was patterned onto an ITO substrate modified with poly(ethylene) glycol (PEG) silane. The photoresist served as a stencil during exposure of the surface to oxygen plasma. Upon incubation with collagen (I) solution and removal of the photoresist, the ITO substrate contained collagen regions surrounded by nonfouling PEG silane. Chemical analysis carried out with time-of-flight secondary ion mass spectrometry (ToF-SIMS) at different stages in micropatterned construction verified removal of PEG-silane during oxygen plasma and presence of collagen and PEG molecules on the same surface. Imaging ellipsometry and atomic force microscopy (AFM) were employed to further investigate micropatterned ITO surfaces. Biological application of this micropatterning strategy was demonstrated through selective attachment of mammalian cells on the ITO substrate. Importantly, after seeding the first cell type, the ITO surfaces could be activated by applying negative voltage (−1.4 V vs Ag/AgCl). This resulted in removal of nonfouling PEG layer and allowed to attach another cell type onto the same surface and to create micropatterned cocultures. Micropatterned cocultures of primary hepatocytes and fibroblasts created by this strategy remained functional after 9 days as verified by analysis of hepatic albumin. The novel surface engineering strategy described here may be used to pattern multiple cell types on an optically transparent and conductive substrate and is envisioned to have applications in tissue engineering and biosensing.

DOI:10.1021/am900508m

The ability to direct proliferation and growth of living cells using chemically and topologically textured surfaces is finding many niche applications, both in fundamental biophysical investigations of cell-surface attachment and in developing design principles for many tissue engineering applications. Here we address cellular adhesion behavior on solid patterns of differing wettability (a static substrate) and fluid patterns of membrane topology (a dynamic substrate). We find striking differences in the cellular adhesion characteristics of lipid mono- and bilayers, despite their essentially identical surface chemical and structural character. These differences point to the importance of subtle variations in the physical properties of the lipid mono- and bilayers (e.g., membrane tension and out-of-plane undulations). Furthermore, we find that introducing phosphatidylserine into the patterned lipidic substrates causes a loss of cell-patterning capability. Implications of this finding for the mechanism by which phosphatidylserine promotes cellular adhesion are discussed.

DOI:10.1021/la900166u

In this paper we describe a microfabrication-derived approach for defining interactions between distinct groups of cells and integrating biosensors with cellular micropatterns. In this approach, photoresist lithography was employed to micropattern cell-adhesive ligand (collagen I) on silane-modified glass substrates. Poly(ethylene glycol) (PEG) photolithography was then used to fabricate hydrogel microstructures in registration with existing collagen I domains. A glass substrate modified in this manner had three types of micrpatterned regions: cell-adhesive collagen I domains, moderately adhesive silanized glass regions, and nonadhesive PEG hydrogel regions. Incubation of this substrate with primary rat hepatocytes or HepG2 cells resulted in attachment of hepatic cells on collagen I domains with no adhesion observed on silane-modified glass regions or hydrogel domains. 3T3 fibroblasts added onto the same surface attached on the glass regions around the hepatocytes, completing the coculture. Significantly, PEG hydrogel microstructures remained free of cells and were used to “fence” hepatocytes from fibroblasts, thus limiting communication between the cell types. We also demonstrated that entrapment of enzyme molecules inside hydrogel microstructures did not compromise nonfouling properties of PEG. Building on this result, horse radish peroxidase-containing hydrogel microstructures were integrated into micropatterned cocultures and were used to detect hydrogen peroxide in the culture medium. The surface micropatterning approach described here may be used in the future to simultaneously define and detect endocrine signaling between two distinct cell types.

DOI:10.1021/la803635r

Neutron reflectometry was used to probe in situ the structure of supported lipid bilayers at the solid−liquid interface during the early stages of UV-induced oxidative degradation. Single-component supported lipid bilayers composed of gel phase, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and fluid phase, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), phospholipids were exposed to low-dose oxidative stress generated by UV light and their structures were examined by neutron reflectometry. An interrupted illumination mode, involving exposures in 15 min increments with 2 h intervals between subsequent exposures, and a continuous mode involving a single 60 (or 90) min exposure period were employed. In both cases, pronounced differences in the structure of the lipid bilayer after exposure were observed. Interrupted exposure led to a substantial decrease in membrane coverage but preserved its total thickness at reduced scattering length densities. These results indicate that the initial phase during UV-induced membrane degradation involves the formation of hydrophilic channels within the membrane. This is consistent with the loss of some lipid molecules we observe and attendant reorganization of residual lipids forming hemimicellar edges of the hydrophilic channels. In contrast, continuous illumination produced a graded interface of continuously varied scattering length density (and hence hydrocarbon density) extending 100−150 Å into the liquid phase. Exposure of a DPPC bilayer to UV light in the presence of a reservoir of unfused vesicles showed low net membrane disintegration during oxidative stress, presumably because of surface back-filling from the bulk reservoir. Chemical evidence for membrane degradation was obtained by mass spectrometry and Fourier transform infrared spectroscopy. Further evidence for the formation of hydrophilic channels was furnished by fluorescence microscopy and imaging ellipsometry data.

DOI:10.1021/ja807680m

2008

The asymmetric distribution of charged molecules between the leaflets of solid-substrate-supported phospholipid bilayers is studied using imaging ellipsometry, fluorescence microscopy, and numerical solutions of the Poisson−Boltzmann equation. Experiments are facilitated by the use of patterned substrates that allow for side-by-side comparison of lipid monolayers and supported bilayers. On silica surfaces, negatively charged lipid components are shown to be enriched in the outer leaflet of a supported bilayer system at modest salt concentrations. The approaches developed provide a general means for determining asymmetries of charged components in supported lipid bilayers.

DOI:10.1021/la802909c

We show that a two-step process, involving spontaneous self-assembly of lipids and apolipoproteins and surface patterning, produces single, supported lipid bilayers over two discrete and independently adjustable length scales. Specifically, an aqueous phase incubation of DMPC vesicles with purified apolipoprotein A-I results in the reconstitution of high density lipoprotein (rHDL), wherein nanoscale clusters of single lipid bilayers are corralled by the protein. Adsorption of these discoidal particles to clean hydrophilic glass (or silicon) followed by direct exposure to a spatial pattern of short-wavelength UV radiation directly produces microscopic patterns of nanostructured bilayers. Alternatively, simple incubation of aqueous phase rHDL with a chemically patterned hydrophilic/hydrophobic surface produces a novel compositional pattern, caused by an increased affinity for adsorption onto hydrophilic regions relative to the surrounding hydrophobic regions. Further, by simple chemical denaturation of the boundary protein, nanoscale compartmentalization can be selectively erased, thus producing patterns of laterally fluid, lipid bilayers structured solely at the mesoscopic length scale. Since these aqueous phase microarrays of nanostructured lipid bilayers allow for membrane proteins to be embedded within single nanoscale bilayer compartments, they present a viable means of generating high-density membrane protein arrays. Such a system would permit in-depth elucidation of membrane protein structure−function relationships and the consequences of membrane compartmentalization on lipid dynamics.

DOI:10.1021/ja803110v

Triglyceride-rich lipoprotein (TGRL) lipolysis may provide a proinflammatory stimulus to endothelium. Detergent-resistant plasma membrane microdomains (lipid rafts) have a number of functions in endothelial cell inflammation. The mechanisms of TGRL lipolysis-induced endothelial cell injury were investigated by examining endothelial cell lipid rafts and production of reactive oxygen species (ROS). Lipid raft microdomains in human aortic endothelial cells were visualized by confocal microscopy with fluorescein isothiocyanate-labeled cholera toxin B as a lipid raft marker. Incubation of Atto565-labeled TGRL with lipid raft-labeled endothelial cells showed that TGRL colocalized with the lipid rafts, TGRL lipolysis caused clustering and aggregation of lipid rafts, and colocalization of TGRL remnant particles on the endothelial cells aggregated lipid rafts. Furthermore, TGRL lipolysis caused translocation of low-density lipoprotein receptor-related protein, endothelial nitric oxide synthase, and caveolin-1 from raft regions to nonraft regions of the membrane 3 h after treatment with TGRL lipolysis. TGRL lipolysis significantly increased the production of ROS in endothelial cells, and both NADPH oxidase and cytochrome P-450 inhibitors reduced production of ROS. Our studies suggest that alteration of lipid raft morphology and composition and ROS production could contribute to TGRL lipolysis-mediated endothelial cell injury.

DOI:10.1152/ajpheart.01366.2007

Via imaging ellipsometry, we study the phase transition dynamics induced by selective gelation of one component in a binary supported phopholipid bilayer. We find the modulation of two attendant morphological features: emergence of extended defect chains due to a net change in the molecular areas and fractal-like domains suggesting weak line tension. A time-lapse analysis of the ellipsometric images reveals the cluster size of 4–20 molecules undergoing gelation indicating weak cooperativity. These results demonstrate the use of ellipsometry for in situ, label-free, non-contact, and large-area imaging of dynamics in interfacial films.

DOI:10.1039/B801390J

Disaccharides are known to protect sensitive biomolecules against stresses caused by dehydration, both in vivo and in vitro. Here we demonstrate how interfacial accumulation of trehalose can be used to (1) produce rugged supported lipid bilayers capable of near total dehydration; (2) enable spatial patterning of membrane micro-arrays; and (3) form stable bilayers on otherwise lipophobic substrates (e.g., metal transducers) thus affording protecting, patterning, and scaffolding of lipid bilayers.

DOI:10.1039/B800370J

The surface-assisted fusion, rupture, and spreading of vesicles and hydration-induced spreading of lipids onto chemically and topographically structured surfaces gives rise to lipid structures useful for modeling many physical-chemical properties of lipid bilayers. Chemically structured surfaces produce a lipid structure revealing template-induced assembly of coexisting lipid phases, which reflect the underlying pattern of surface energy, wettability, and chemistry. In a construct derived using photochemically patterned molecular monolayers, the author found a spontaneous separation of fluid bilayer regions from the fluid monolayer regions by a controllable transition region or moat. The coexisting bilayer/monolayer morphologies derived from single vesicular sources are particularly attractive for the study of a range of leaflet-dependent biophysical phenomena and offer a new self-assembly strategy for synthesizing large-scale arrays of functional bilayer specific substructures including ion-channels and membrane-proteins. The uses of topologically patterned surfaces similarly provide new models to design complex three-dimensional membrane topographies and curvatures. These platforms promise fundamental biophysical studies of curvature-dependent membrane processes as well as useful bioanalytical devices for molecular separations within fluid amphiphilic membrane environments. Some future directions enabled by lipid self-assembly at structured surfaces are also discussed.

DOI:10.1116/1.2889055

We combine hierarchical surface wrinkling of elastomers with lipid membrane deposition techniques to dynamically template complex three-dimensional topographies onto supported lipid bilayers. The real-time introduction of corresponding nano- to micrometer scale curvatures triggers spatially periodic, elastic bending of the bilayer, accompanied by molecular-level reorganizations. This ability to dynamically impose curvatures on supported bilayers and the ensuing re-equilibration promises fundamental material and biophysical investigations of curvature-dependent, static heterogeneities and dynamic reorganizations pervasive in biological membranes.

DOI:10.1021/nl073085b

Patterning physical, chemical, and biological functions at solid surfaces combines technological development with scientific discoveries in many disparate fields. A variety of top-down and bottom-up approaches has proved successful for applications in the solid state, affording large-area patterning at ever-shrinking length scales. Here we review a collection of recent efforts that highlight the versatility of short-wavelength ultraviolet light and photogenerated reactive oxygen species as a simple and cost-effective means to pattern a variety of challenging materials and thin-film configurations. In particular, we discuss two different classes of materials that present different challenges for patterning: fluid phospholipid bilayers at the buried solid-water interface and the surfaces of bulk elastomers. Despite the use of an identical patterning source, the generation and stabilization of patterns in these two classes of materials follow different mechanisms and produce different functionalities.

DOI:10.1146/annurev.physchem.58.032806.104644

2007

A simple integration of molecular and colloidal self-assembly approaches with photopatterning is shown to produce multifunctional patterns of amphiphilic colloidal crystals. These crystals display binary spatial patterns of wettability by water and a single photonic stop-band in air. Upon exposure to water, the uniform stop-band is replaced by a pattern of coexisting stop-bands that reflect the underlying pattern of surface wetting. These hydration-dependent photonic patterns within single colloidal crystals form because of near-complete water rejection from the three-dimensionally disposed nanoscale interstices in hydrophobic regions and its exclusive permeation within the hydrophilic regions. This water permeation pattern is further structured by the three-dimensional (3D) distribution and contiguity of the nanoscale interstices between individual colloids, allowing 3D patterned organization of functional units in secondary self-assembly processes, as illustrated using quantum dots, metal nanoparticles, and fluorescent probes.

DOI:10.1021/nl072483b

The integration of ion-channel transport functions with responses derived from nanostructured and nanoporous silica mesophase materials is demonstrated. Patterned thin-film mesophases consisting of alternating hydrophilic nanoporous regions and hydrophobic nanostructured regions allow for spatially localized proton transport via selective dimerization of gramicidin in lipid bilayers formed on the hydrophilic regions. The adjoining hydrophobic mesostructure doped with a pH sensitive dye reports the transport. The ease of integrating functional membranes and reporters through the use of patterned mesophases should enable high throughput studies of membrane transport.

DOI:10.1021/nl071184j

This article describes the fluorescence microscopy and imaging ellipsometry-based characterization of supported phospholipid bilayer formation on elastomeric substrates and its application in microcontact printing of spatially patterned phospholipid bilayers. Elastomeric stamps, displaying a uniformly spaced array of square wells (20, 50, and 100 μm linear dimensions), are prepared using poly(dimethyl)siloxane from photolithographically derived silicon masters. Exposing elastomeric stamps, following UV/ozone-induced oxidation, to a solution of small unilamellar phospholipid vesicles results in the formation of a 2D contiguous, fluid phospholipid bilayers. The bilayer covers both the elevated and depressed regions of the stamp and exhibits a lateral connectivity allowing molecular transport across the topographic boundaries. Applications of these bilayer-coated elastomeric stamps in microcontact printing of lipid bilayers reveal a fluid-tearing process wherein the bilayer in contact regions selectively transfers with 75−90% efficiency, leaving behind unperturbed patches in the depressed regions of the stamp. Next, using cholera-toxin binding fluid POPC bilayers that have been asymmetrically doped with ganglioside Gm1 ligand in the outer leaflets, we examine whether the microcontact transfer of bilayers results in the inversion of the lipid leaflets. Our results suggest a complex transfer process involving at least partial bilayer reorganization and molecular re-equilibration during (or upon) substrate transfer. Taken together, the study sheds light on the structuring of lipid inks on PDMS elastomers and provides clues regarding the mechanism of bilayer transfer. It further highlights some important differences in stamping fluid bilayers from the more routine applications of stamping in the creation of patterned self-assembled monolayers.

DOI:10.1021/la701920v

Spontaneous spreading of phospholipids following hydration by water is directly compared on hydrophilic and hydrophobic surfaces using planar, composite substrates exhibiting binary patterns of surface energies. The use of patterned substrates—in conjunction with real-time, quantitative applications of imaging ellipsometry and epifluorescence microscopy—affords a side-by-side comparison of spreading kinetics and equilibrium morphologies following the hydration of a single parent-lipid source. We find that for fluid phospholipids (T > Tm), substantially contiguous bimolecular and mono-molecular lipid layers spread away from a source on hydrophilic and hydrophobic surfaces, respectively. For bilayers and monolayers, the advancing lipid sheets exhibit square-root-of-time dependent kinetics reflecting a balance between a spreading force and a resistive drag. Furthermore, monolayers advance at only a slightly faster rate (by a factor of 1.7 ± 0.2) than bilayers despite a substantially higher available spreading energy. This apparently counter-intuitive observation can be reconciled in terms of a corresponding rise in the competing drag energy—consistent with the differences in slip-planes associated with mono- and bilayer spreading.

DOI:10.1039/B704827K

A direct illumination by focused femtosecond pulses from a near-infrared Ti:sapphire laser in nanojoules peak-energy regime results in a highly localized removal of phospholipids from a fluid, supported lipid bilayer submerged in aqueous media. Lipid-free gaps created in this manner exhibit long-term stability and interrupt membrane fluidity, thereby providing diffusional barriers within the membrane environment. Re-illuminating these lipid-free barriers at comparable peak energies but at higher repetition (∼200×) rapidly and selectively erases them. This in situ, reversible, maskless, multiphoton membrane photolithography provides a new means to compartmentalize and regulate membrane fluidity by erecting and erasing diffusional barriers at time scales faster than diffusional time scales. Such an ability to dynamically manipulate membrane diffusional properties should prove useful in designing synthetic models for studies of thermodynamically uphill processes including directed-molecular transport, compositional and free-energy gradients, and nonequilibrium fluctuations in biological membranes.

DOI:10.1021/ja065626p

Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.

DOI:10.1529/biophysj.106.097071

2006

Using a combination of fluorescence correlation and infrared absorption spectroscopies, we characterize lipid lateral diffusion and membrane phase structure as a function of protein binding to the membrane surface. In a supported membrane configuration, cholera toxin binding to the pentasaccharaide headgroup of membrane-incorporated GM1 lipid alters the long-range lateral diffusion of fluorescently labeled probe lipids, which are not involved in the binding interaction. This effect is prominently amplified near the gel-fluid transition temperature, Tm, of the majority lipid component. At temperatures near Tm, large changes in probe lipid diffusion are measured at average protein coverage densities as low as 0.02 area fraction. Spectral shifts of the methylene symmetric and asymmetric stretching modes in the lipid acyl chain confirm that protein binding alters the fraction of lipid in the gel phase.

DOI:10.1021/ja064093h

Probing membranes: Nonspecific interactions between glass beads and supported lipid membranes act as a simple tool to characterize their local structure and mechanical properties. The beads differentiate monolayers (see figure B) from fluid bilayers (A) by inducing a qualitatively different elastic response, characterized by undeformed binding for monolayers and membrane wrapping for bilayers.

DOI:10.1002/cphc.200600222

Supported membranes represent an elegant route to designing well-defined fluid interfaces which mimic many physical–chemical properties of biological membranes. Recent years have witnessed rapid growth in the applications of physical and materials science approaches in understanding and controlling lipid membranes. Applying these approaches is enabling the determination of their structure–dynamics–function relations and allowing the design of membrane-mimetic devices. The collection of articles presented in this issue of MRS Bulletin illustrates the breadth of activity in this growing partnership between materials science and biophysics. Together, these articles highlight some of the key challenges of cellular membranes and exemplify their utility in fundamental biophysical studies and technological applications. The topics covered also confirm the importance of lipid membranes as an exciting example of soft condensed matter. We hope that this issue will serve readers by highlighting the intellectual scope and emerging opportunities in this highly interdisciplinary area of ma- terials research.

DOI:10.1557/mrs2006.134

We have developed a simple method to introduce cholesterol- and sphingomyelin-rich chemical heterogeneities into controlled densities and concentrations within predetermined regions of another distinct fluid phospholipid bilayer supported on a solid substrate. A contiguous primary phase--a fluid POPC bilayer displaying a well-defined array of lipid-free voids (e.g., 20-100 microm squares)--was first prepared on a clean glass surface by microcontact printing under water using a poly(dimethylsiloxane) stamp. The aqueous-phase primary bilayer pattern was subsequently incubated with secondary-phase small unilamellar vesicles composed of independent chemical compositions. Backfilling by comparable vesicles resulted in gradual mixing between the primary- and secondary-phase lipids, effacing the pattern. When the secondary vesicles consisted of phase-separating mixtures of cholesterol, sphingomyelin, and a phospholipid (2:1:1 POPC/sphingomyelin/cholesterol or 1:1:1 DOPC/sphingomyelin/cholesterol), well-defined spatial patterns of fluorescence, chemical compositions, and fluidities emerged. We conjecture that these patterns form because of the differences in the equilibration rates of the secondary liquid-ordered and liquid-disordered phases with the primary fluid POPC phase. The pattern stability depended strongly on the ambient-phase temperature, cholesterol concentration, and miscibility contrast between the two phases. When cholesterol concentration in the secondary vesicles was below 20 mol %, secondary intercalants gradually diffused within the primary POPC bilayer phase, ultimately dissolving the pattern in several minutes and presumably forming a new quasi-equilibrated lipid mixture. These phase domain micropatterns retain some properties of biological rafts including detergent resistance and phase mixing induced by selective cholesterol extraction. These patterns enable direct comparisons of cholesterol- and sphingomyelin-rich phase domains and fluid phospholipid phases for their functional preferences and may be useful for developing simple, parallelized assays for phase and chemical composition-dependent membrane functionalities.

DOI:10.1021/la052248d

purpose. To investigate whether the signaling events occurring in Fas-mediated apoptosis alter raft membrane formation in human RPE cells. methods. Formation of lipid rafts in cultured human retinal pigment epithelial cells (ARPE-19) was studied by confocal microscopy, with fluorescein-labeled cholera toxin subunit B binding protein (BODIPY)–labeled ganglioside GM1 lipid after Fas-L induction of apoptosis. Apoptosis was assessed by fluorescein-labeled annexin V detection of phosphatidylserine externalization and quadrant analysis with flow cytometry. Membrane rafts were localized into membrane vesicles by passing BODIPY-labeled GM1 RPE cells through a 2-μm-pore polycarbonate membrane using an extruder device. The labeled fractions, containing vesicles enriched in GM1, were detected by flow cytometry and then analyzed for the presence of Fas protein. results. Differential punctate staining of membrane rafts was demonstrated in normal and FasL-induced apoptotic human ARPE-19 cells in culture by confocal microscopy, using cholera toxin B and GM1 labeling of extruded vesicles. The lipid raft–associated vesicles were derived by plasma membrane dissociation, via a newly developed whole-cell extrusion technique that produced 2-μm vesicles with both GM1 lipid and Fas protein abundance enriched in a subpopulation of the membrane-derived vesicles. The amount of Fas protein in the vesicles containing raft domains markedly increased in FasL-treated cells. Treatment of human ARPE 19 cells with methyl β-cyclodextrin after FasL induction of apoptosis resulted in cellular cholesterol depletion and markedly reduced the incidence of Fas-receptor localization in GM1 rafts. conclusions. Human ARPE-19 cells in culture contain membrane rafts with apoptotic signaling effectors uniformly distributed in the native state. The cells stimulated to undergo apoptosis appear to use membrane rafts in the death-signaling process by mobilization of rafts to localized regions of the membrane that are now enriched with apoptotic signaling effectors. Fas signaling induces apoptotic raft formation that results in polar condensation, or capping, of the rafts in the late stages of apoptosis. A novel extrusion technique is described that allows localization and enrichment of rafts into membrane vesicles, which can be assayed by flow cytometry. Cholesterol depletion, after Fas ligand activation of apoptosis, reduced raft formation in cells induced to undergo apoptosis. Therapeutic implications for the treatment of retinal disorders are discussed.

DOI:10.1167/iovs.05-1167

We report the formation of a new class of supported membranes consisting of a fluid phospholipid bilayer coupled directly to a broadly tunable colloidal crystal with a well-defined photonic band gap. For nanoscale colloidal crystals exhibiting a band gap at the optical frequencies, substrate-induced vesicle fusion gives rise to a surface bilayer riding onto the crystal surface. The bilayer is two-dimensionally continuous, spanning multiple beads with lateral mobilities which reflect the coupling between the bilayer topography and the curvature of the supporting colloidal surface. In contrast, the spreading of vesicles on micrometer scale colloidal crystals results in the formation of bilayers wrapping individual colloidal beads. We show that simple UV photolithography of colloidal crystals produces binary patterns of crystal wettabilities, photonic stopbands, and corresponding patterns of lipid mono- and bilayer morphologies. We envisage that these approaches will be exploitable for the development of optical transduction assays and microarrays for many membrane-mediated processes, including transport and receptor−ligand interactions.

DOI:10.1021/ja056701j

2005

We report the formation of microscopic patterns of substrate-supported, 3D planar colloidal crystals using physical confinement in conjunction with surfaces displaying predetermined binary patterns of hydropholicity. The formation process involves a primary self-assembly wherein nano- and microscale colloids order into a photonic fcc lattice via capillary interactions followed by a secondary template-induced crystal cleavage step. Following this method, arbitrary arrays of pattern elements, which preserve structural and orientational properties of the parent crystal, can be easily obtained.

DOI:10.1021/la0521692

This paper describes an application of a non-thermal, photochemical calcination process for an efficient and spatially controlled removal of the organic structure-directing agent in the preparation of thin films of microporous or zeolite materials. We prepared thin-films of a high silica zeolite (structure code: MFI) following a previously published procedure. The films were illuminated using an ozone generating short-wavelength ultraviolet light in ambient environments and characterized using Fourier-transform infrared spectroscopy, imaging ellipsometry, thin-film X-ray diffraction, and scanning electron microscopy. Results presented here indicate that the UV/ozone treatment under nominally room temperature conditions leads to complete removal of template (structure-directing-agent) from zeolite films comparable to that achieved by thermal calcination. Furthermore, spatially addressing the UV/ozone illumination pattern using a physical mask resulted in the lateral confinement of the template removal from the zeolite film leaving behind a composite film composed of templated and template-free regions. Subsequent chemical treatment of the patterned film selectively removed the as-synthesized, unexposed, regions of the film thereby providing a means for the creation of isolated zeolite film islands at predetermined locations on the substrate surface.

DOI:10.1016/j.micromeso.2005.07.048

We show that two dips of an oxidized silicon substrate through a prepolymerized n-octadecylsiloxane monolayer at an air−water interface in a rapid succession produces periodic, linear striped patterns in film morphology extending over macroscopic area of the substrate surface. Langmuir monolayers of n-octadecyltrimethoxysilane were prepared at the surface of an acidic subphase (pH 2) maintained at room temperature (22 ± 2 °C) under relative humidities of 50−70%. The substrate was first withdrawn at a high dipping rate from the quiescent aqueous subphase (upstroke) maintained at several surface pressures corresponding to a condensed monolayer state and lowered soon after at the same rate into the monolayer covered subphase (downstroke). The film structure and morphology were characterized using a combination of optical microscopy, imaging ellipsometry, and Fourier transform infrared spectroscopy. An extended striped pattern, perpendicular to the pushing direction of the second stroke, resulted for all surface pressures when the dipping rate exceeded a threshold value of 40 mm min-1. Below this threshold value, uniform deposition characterizing formation of a bimolecular film was obtained. Under conditions that favored striped deposition during the downstroke through the monolayer-covered interface, we observed a periodic auto-oscillatory behavior of the meniscus. The stripes appear to be formed by a highly correlated reorganization and/or exchange of the first monolayer, mediated by the Langmuir monolayer at the air−water interface. This mechanism appears distinctly different from nanometer scale stripes observed recently in single transfers of phospholipid monolayers maintained near a phase boundary. The stripes further exhibit wettability patterns useful for spatially selective functionalization, as demonstrated by directed adsorptions of an organic dye (fluorescein) and an oil (hexadecane).

DOI:10.1021/la050735n

The energetics of silver alkanethiolates with various long-chain lengths, formed through stepwise hierarchical assembly involving primary directional interactions between Ag and S forming the inorganic core and secondary stacking facilitating the formation of the three-dimensional structure, were directly assessed by in situ synthesis calorimetry. The thermal evolution of the materials is discussed in terms of confinement and synergy between enthalpy and entropy factors.

DOI:10.1021/cm050961i

Single bilayer membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were formed on ordered nanocomposite and nanoporous silica thin films by fusion of small unilamellar vesicles. The structure of these membranes was investigated using neutron reflectivity. The underlying thin films were formed by evaporation induced self-assembly to obtain periodic arrangements of silica and surfactant molecules in the nanocomposite thin films, followed by photocalcination to oxidatively remove the organics and render the films nanoporous. We show that this platform affords homogeneous and continuous bilayer membranes that have promising applications as model membranes and sensors.

DOI:10.1021/la0471240

Holographic gratings are recorded in azo-dye nitrobenzoxazole-labeled phospholipid thin films by use of 244-nm UV light. The gratings continue to grow for more than 1 h, even after the recording light is removed. The diffraction efficiency of these gratings shows extreme sensitivity to humidity and can increase reversibly by 2 orders of magnitude in air that is saturated with water vapor. This effect is related to the unique characteristics of phospholipid molecules that undergo hydration-dependent structural reorganization and self-assembly.

DOI:10.1364/OL.30.000501

A lithographic method of patterning lipid bilayer membranes on the surface of colloidal particles is described. Three-dimensional, micrometer-resolution patterns on the single fluid lipid bilayer membranes supported on silica microspheres have been generated by in-situ UV photolithography (see Figure). This technique is based on the direct photochemical removal of lipids from the colloid surface in an aqueous environment.

DOI:10.1002/adma.200401586

The evolution of photochemical surfactant removal and silica condensation from organically templated thin film silica nanocomposites with mesoscopic ordering has been probed using a combined application of Fourier transform infrared (FT-IR) spectroscopy and single wavelength ellipsometry. Thin films of silica nanocomposites were prepared by a previously reported evaporation-induced self-assembly process. Specifically, oxidized silicon and gold substrates were withdrawn at 25 mm/min from a subcritical micelle concentration solution containing an ethylene oxide surfactant as a structure-directing agent and tetraethyl orthosilicate as a silica precursor. Real-time grazing incidence difference FT-IR spectra of the nanocomposite films on gold taken during exposure to short-wavelength ultraviolet light (184−257 nm) show that surfactant removal and silica condensation occur gradually and concomitantly. Surfactant removal and silica reconstructions were found to be nearly complete after 90 min of exposure. Further, a transient feature was observed in the FT-IR spectra around 1713 cm-1 during the UV exposure process and was assigned to a carbonyl (CO) stretching mode absorption, reflecting the transient formation of a partially oxidized surfactant intermediate. From these data we propose a stepwise model for surfactant removal from the nanocomposite films. Ellipsometrically determined index of refraction values collected as a function of UV exposure are also shown to support such a stepwise mechanism of surfactant removal from the ordered nanocomposite silica thin film mesophases studied here.

DOI:0.1021/jp051401+

We have studied the spreading of phospholipid vesicles on photochemically patterned n-octadecylsiloxane monolayers using epifluorescence and imaging ellipsometry measurements. Self-assembled monolayers of n-octadecylsiloxanes were patterned using short-wavelength ultraviolet radiation and a photomask to produce periodic arrays of patterned hydrophilic domains separated from hydrophobic surroundings. Exposing these patterned surfaces to a solution of small unilamellar vesicles of phospholipids and their mixtures resulted in a complex lipid layer morphology epitaxially reflecting the underlying pattern of hydrophilicity. The hydrophilic square regions of the photopatterned OTS monolayer reflected lipid bilayer formation, and the hydrophobic OTS residues supported lipid monolayers. We further observed the existence of a boundary region composed of a nonfluid lipid phase and a lipid-free moat at the interface between the lipid monolayer and bilayer morphologies spontaneously corralling the fluid bilayers. The outer-edge of the boundary region was found to be accessible for subsequent adsorption by proteins (e.g., streptavidin and BSA), but the inner-edge closer to the bilayer remained resistant to adsorption by protein or vesicles. Mechanistic implications of our results in terms of the effects of substrate topochemical character are discussed. Furthermore, our results provide a basis for the construction of complex biomembrane models, which exhibit fluidity barriers and differentiate membrane properties based on correspondence between lipid leaflets. We also envisage the use of this construct where two-dimensionally fluid, low-defect lipid layers serve as sacrificial resists for the deposition of protein and other material patterns.

DOI:10.1021/ja043439q

2004

A wet photolithographic route for micropatterning fluid phospholipid bilayers is demonstrated in which spatially directed illumination by short-wavelength ultraviolet radiation results in highly localized photochemical degradation of the exposed lipids. Using this method, we can directly engineer patterns of hydrophilic voids within a fluid membrane as well as isolated membrane corrals over large substrate areas. We show that the lipid-free regions can be refilled by the same or other lipids and lipid mixtures which establish contiguity with the existing membrane, thereby providing a synthetic means for manipulating membrane compositions, engineering metastable membrane microdomains, probing 2D lipid−lipid mixing, and designing membrane-embedded arrays of soluble proteins. Following this route, new constructs can be envisaged for high-throughput membrane proteomic, biosensor array, and spatially directed, aqueous-phase material synthesis.

DOI:10.1021/ja047714k

A wet photolithography approach using light-activated, localized, oxidative chemistry can directly pattern fluid phospholipid bilayers submerged in aqueous phases. Targeted incorporation of secondary components within pattern voids (see Figure) allows many membrane dynamical processes to be probed and optically defined arrays of holes, functional membrane microdomains, and proteins embedded in a lipidic background can be designed.

DOI:10.1002/adma.200306498

The binding properties of cholera toxin B (CTB) oligomer to substrate supported membrane bilayer, containing physiologically relevant concentrations of receptor glycolipids, viz. monosialoganglioside (GM1), have been extensively studied by the atomic force microscopy (AFM). Two distinct classes of GM1 containing membrane-mimetic surfaces were prepared: supported lipid bilayer membranes (sBLMs) on freshly cleaved mica and hybrid lipid bilayer membranes (hBLMs) on octadecyltrichlorosilane (OTS) derivatized silicon substrates. On sBLMs, aggregates with a well-defined ordered arrangement of individual CTB molecules were observed at all GM1 and cholera concentrations studied. In sharp contrast, features consistent with randomly distributed adsorbed individual CTB molecules were seen on a bare mica surface. On the hBLMs, the aggregate structures were only observed when the bilayer was formed onto ordered OTS surfaces, offering continuous and defect-free lipid membrane for the lateral diffusion of GM1. Ill-packed and disordered OTS monolayers yielded a random distribution of adsorbed proteins comparable to that observed for CTB binding on bare mica substrates. These observations strongly support that the aggregation of CTB-GM1 complex is a result of the specific interaction of CTB molecules with GM1 receptors in the fluid membrane bilayers. The high mobility of GM1 allows lateral diffusion of the complex to form ordered aggregates.

DOI:10.1016/j.colsurfb.2003.09.001

2003

This special issue, The Biomolecular Interface, addresses an area of study that defines the field of biological surface science. This field, which has evolved naturally from the study of soft organic matter and the chemistry and physics of self-assembly, represents a fusion of two disciplines, materials science and biological science. Each of these highly multidisciplinary fields is experiencing explosive intellectual and technological progress. The tools for study of this new field include surface science techniques such as scanned probe microscopies and molecular biology strategies such as genetic engineering.

DOI:10.1021/la034035z

We have studied the effects of relative mole ratios of the reactant precursors in the one-phase synthesis of alkaneselenoate- and alkanethiolate-functionalized gold nanoparticles. Specifically, we prepared a series of dodecaneselenoate (DDSe)- and dodecanethiolate (DDT)-functionalized gold nanoparticles using four different Se/Au and S/Au mole ratios in reactant mixtures at two different reaction temperatures employing three different solvents. In all cases, the synthesis relied on the reduction of H[AuCl4], in the presence of dodecanethiol (DDT) and didodecyl diselenide (DD2Se2) using lithium triethylborohydride (superhydride) as the reducing agent. Nanoparticle formation, structure, and bonding characteristics were investigated using a combination of transmission electron microscopy, UV absorption spectroscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. Passivation by alkyl selenide was more efficient and was characterized by greater chain density and stronger Au−Se bond strength when high ligand/substrate ratios were employed. Particle size was surprisingly uniform in all cases, independent of mole ratio. By contrast, particle size (2−5 nm) was found to increase with increasing mole ratios when the passivating ligand was alkanethiolate, whose chain grafting density increased with increasing mole ratio, fully coincided with the literature. These results can be reconciled in terms of a simple mechanistic scenario wherein the nanoparticle formation using alkanethiolate ligands proceeds via the formation of a “polymer-like” intermediate between the Au ions and the alkanethiolate ligands prior to reduction whereas such an intermediate is not formed when selenoate is used as the binding ligand.

DOI:10.1021/la020628i

We present a simple molecular model for the self-assembly of alkanethiols on gold. The model, a rigid rod representation of a molecule which is constrained to a given distance from the gold surface, allows direct long-time simulations of large-scale molecular ensembles (104−105 molecules) on desktop workstations. As a result, the model allows for efficient studies of evolution and ordering of, for example, orientational order and domain patterns in a full range of monolayer coverages. The model is parametrized entirely by existing literature on atomic and molecular scale interactions. Extensive simulations of molecular self-assembled monolayer domain formation at optimal (packing commensurate with a gold surface (111) structure) and suboptimal packing are conducted and presented. The results show close correspondence between the model features and the existing, and emerging, picture observed through experimental characterization of self-assembled monolayers on Au(111). This strong experimental validation of the conformationally insensitive molecular model suggests that the conformational degrees of freedom are not essential for the self-assembly of alkanethiols on gold. It appears that the interplay between the substrate−headgroup and chain−chain interactions determines the self-assembly process and the emergent molecular structures. The presentation of simulation results for different molecular surface coverages is used to derive a primitive two-dimensional isothermal phase diagram. The latter was found to be in good general agreement with available experimental data and provides insight into the formation and growth mechanism of monolayers. The work suggests directions for a minimal approach to studying at least some of the complexity contained in molecular self-assembly processes.

DOI:10.1021/la026344f

Here we present a spatially directed calcination approach based on masked UV exposure to pattern mesoporous regions within a mesostructured matrix in a rapid, single-step, and inexpensive manner. Subsequent chemical treatment of the film can selectively remove the mesostructured regions, leading to patterned mesoporous structures. Such tunability in the processing under near room-temperature conditions allows for spatial control and patterning of function related to optical properties, topology, porosity, hydrophobicity, and structural morphology of the mesoscopic thin film material on a wide range of substrates.

DOI:10.1021/nl0341279

2002

Multilayer films comprised of a generation 3.0 dendrimer and NLO-active polyanion PAZO were formed by ionic self-assembly. The films were studied by UV−visible spectroscopy, single-wavelength ellipsometry, and second-harmonic generation. We show that the multilayer formation mechanism involves an initial adsorption of the polymer that reaches saturation after 5 min, while conformational changes equilibrate after 10 min. The terminal layer is more loosely packed than the nonterminal layers that have interpenetrated zones containing both the dendrimer and PAZO. Most of the anisotropic order of the NLO-active chromophores is thought to exist at the interfacial regions between these layers. From SHG polarization studies, we determine the angle of these ordered PAZO chromophores to be 20.5° ± 5° with respect to the surface normal.

DOI:10.1021/jp012526r

2000

A nominally room temperature photochemical method, simply employing ultraviolet light- (187−254 nm) generated ozone environment, is shown to provide an efficient alternative for the removal of surfactant templates for a routine production of mesoporous silica thin films at low temperatures. The treatment concomitantly strengthens the silicate phase by fostering the condensation of unreacted silanols leading to mesoporous thin films with well-defined mesoscopic morphologies. The surfactant/silicate thin film mesophases were prepared onto a polycrystalline Au surface by dip-coating or spin-casting methods using sub-critical micelle concentration (cmc) nonionic ethylene oxide surfactant in an oligomeric silica sol mixture. The structures and compositions of the thin film mesophases before and after exposure to UV/ozone were determined using a combination of reflection−absorption Fourier transform infrared spectroscopy, transmission electron microscopy, and thin film X-ray diffraction measurements. The pore characteristics of the UV/ozone-treated films were determined using nitrogen adsorption/desporption isotherm measurements. Results presented here clearly establish that the UV/ozone processing leads to complete removal of the surfactant template; strengthens the inorganic skeleton by fostering silica condensation; and renders the mesophase thin film surfaces highly hydrophilic. Two of the most attractive features of the method developed here, namely its usefulness in applications for temperature intolerant substrates (e.g., thin metal films) and in spatially selective removal of the surfactant templates to create patterns of mesoporous thin films, are also illustrated. Finally, the mechanistic implications of these observations are also discussed.

DOI:10.1021/cm000456f

Using temperature-dependent Fourier transform infrared (FTIR) spectroscopy, we probe the molecular level, chain-structural dynamics associated with solid−solid transitions between 25 and 250 °C in a layered inorganic−organic silver dodecanethiolate, AgS(CH2)11CH3. Spectroscopic evidence presented here establishes two major transitions:  the transition occurring at ∼130 °C is characterized by an abrupt, but fully reversible, change in the chain conformational order from an initial all-trans state to the one characterized by mixed or partial chain disorder. The observation of this phase transition is consistent with the previous predictions of a rapid and drastic change in the structural motif from an initial bilayer to the final micellar state. The second transition at about 190 °C, which is consistent with the previous assignment of micellar amorphous transition, is furthermore irreversible and represents thermal degradation of the material. Implications of these results for the general family of chain molecular assemblies in constrained molecular environments are discussed.

DOI:10.1021/jp992156v

1999

Prepolymerized n-octadecyltrichlorosilane (OTS) monolayers were deposited onto oxidized silicon substrates from precursor Langmuir monolayers (at an air−water interface) in two-dimensional liquid expanded (LE), liquid condensed (LC), or mixed (LE/LC coexistence phase) states at four different pulling rates. Morphologies of the transferred monolayers have been investigated using atomic force microscopy (AFM). The OTS monolayers formed from the LE phase precursor reveal an incipient condensation transition exhibiting a novel ring-in-a-ring morphology, wherein uniformly distributed circular domains consisting of two concentric walls of ordered OTS molecules in a high density phase both sandwich and encapsulate disordered OTS molecules in a reduced density phase. On the other hand, the monolayers formed from the LC/LE phase precursor implicate a complete condensation transition, evidenced in the AFM images showing a uniform tiling of near-circular domains composed of ordered OTS molecules in a dense monolayer phase. The monolayers derived from the 2D solid or LC precursor state reveal near-complete surface coverages and uniform film structures, comparable to those obtained by adsorption from a dilute organic solution of OTS molecules (conventional self-assembly process). These structural reconstructions at the substrate surface, namely lateral redistribution into 2D domains, condensation transitions and film coverages, are discussed in terms of the competition between short range and long range interactions. The most dominant effect of increasing pulling rates is the appearance of coalesced domain structures, presumably due to drainage of the water layer at the substrate surface as well as occasional substrate pinning. These results substantiate the idea that templating surface self-assembly of monolayers by using their Langmuir-phase precursors provides a useful alternative to classical solution-phase self-assembly approaches, and affords a wide range of control over film structures and surface morphologies.

DOI:10.1021/jp992030y

Nonlinear second harmonic generation (SHG), second harmonic microscopy (SHM), and infrared spectroscopy are used to determine the structural and optical properties of the Langmuir−Blodgett (LB) monolayer assemblies of NLO-active, 4-eicosyloxo-(E)-stilbazolium iodide (4-EOSI) on a glass substrate. The packing characteristics of the pretransferred interfacial films are determined using π−A isotherm measurements. The molecular coverage of the transferred films is determined by ellipsometry. The films formed on both sides of the glass substrate show substantial second harmonic (SH) conversion from p-polarized 1064 nm fundamental excitation. The SHG and FTIR measurements imply that the single LB layer on glass is composed of oriented clusters of 4-EOSI molecules that are laterally discontinuous. Ordered clusters up to 40 μm in diameter are directly observed using SHM. Subsequent LB transfers using the same 4-EOSI molecule or an amphiphile of comparable chain-length (eicosanoic acid) fill in the unoccupied vacancies in the first layer. The magnitude of the dominant element of the nonlinear susceptibility and the average molecular orientation angle of the chromophore are determined by modeling the characteristic SHG Maker fringes.

DOI:10.1021/la981072v

Successive depositions of precompressed Langmuir monolayers have been shown to allow reproducible formation of air-stable, lipid−alkylsiloxane hybrid bimolecular architectures at oxidic supports. Specifically, prepolymerized Langmuir−Blodgett films of n-octadecylsiloxane (OTS) monolayers on oxided silicon substrates were used as the hydrophobic templates, upon which compressed monolayers of dipalmitoyl-sn1-glycerophosphatidylcholine (DPPC) and monosialogangliosides (Gm1) were deposited from a low-temperature air−water interface by the horizontal deposition method. Structural features of each leaflet of the resultant bimolecular architectures, namely DPPC/OTS/SiO2/Si and Gm1/OTS/SiO2/Si, were characterized using a combined application of infrared spectroscopy, null-ellipsometry, and surface wetting measurements. In both cases, the outer lipid leaflet (DPPC or Gm1) was found to be structurally decoupled with respect to the inner OTS layer. The inner silane layer was composed of essentially untilted (cant angle, θ = 0−10°), all-trans chains at the dense packing of ∼19 Å2/molecule, consistent with the previously reported structure in solution-phase assembled OTS monolayers. The outer DPPC leaflet, however, was found to be composed of collectively tilted (θ = 36°), all-trans acyl chains at the lower chain-packing density (∼26−28 Å2/chain) whereas the outer Gm1 leaflet was concluded to have essentially untilted chains at similarly lower chain-packing densities (∼23−26 Å2/chain) but with the carbohydrate head-groups disposed in a topologically staggered conformation. The structural independence of the two leaflets in the two classes of bilayered architectures examined here confirms the possibility of independently manipulating the molecular structure in each leaflet of supported hybrid bilayers.

DOI:10.1021/la9813679

The first direct characterization of structures of bi-molecular chain assemblies in a self-consistent series of pillared, layered organic−inorganic long-chain silver (n-alkane) thiolates, (AgS(CH2)nCH3; n = 5, 6, 9, 11, 15, and 17), is reported using the combined application of infrared transmission spectroscopy and powder X-ray diffraction. The structural attributes elucidated include quantitative estimates of average chain orientation, chain conformation, chain−chain translational order, interpenetration of the contiguous layers, as well as void characteristics in the chain matrix. The evidence presented here establishes that the layered chain assemblies sandwiched between the inorganic Ag−S backbones in a double-layer arrangement are comprised of an ordered packing of all-trans-extended chains. The average chain in each assembly is oriented vertically away from the quasi-hexagonal Ag−S lattice, in a two-dimensional pseudo-monoclinic arrangement of domains of 60−70 translationally correlated chains. Small interpenetration between the contiguous layers leads to the formation of regularly spaced 1D channels or corridors. The three-dimensional network of 1D channels alternates between the chain layers. All the chain structural characteristics deduced here are in good conformity with those implied in the model proposed earlier by Dance and co-workers. The present results, together with the previous X-ray analysis for comparable short-chain AgSRs, are used to propose a two-step, hierarchical self-assembly mechanism for the formation of silver (n-alkyl) thiolates. It is proposed that the primary self-assembly process involves the organization of Ag+ and RS- species into puckered sheets of quasi-hexagonally symmetric 2D lattices, with the chain substituents extending on each side. The subsequent self-assembly of these 2D building blocks in the third dimension via complementary stacking appears to complete the formation of sandwiched bimolecular chain assemblies.

DOI:10.1021/jp983938b

The structure of microcontact-printed self-assembled monolayers is significant for a number of technological applications as well as for a fundamental understanding of the self-assembly process. The structural differences between printed and solution-deposited monolayers of alkanethiol on Au(111) have been examined utilizing grazing incidence X-ray diffraction and atomic force microscopy. Although the local structures are found to be similar, the domain structure and therefore macroscopic packing of the molecules are found to vary. Surprisingly, printed decanethiol monolayers appear to be significantly better ordered translationally than those formed by standard solution techniques.

DOI:10.1021/la981067r

1998

Self-assembled multilayers of the three modified cyclodextrins (hexakis(2,3-O-hexyl-6-deoxy-6-amino)-α-cyclodextrin (1), heptakis(2,3-O-hexyl-6-deoxy-6-amino)-β-cyclodextrin (2), and octakis(2,3-O-hexyl-6-deoxy-6-amino)-γ-cyclodextrin (3)) on bare gold, as well as on gold surfaces modified with mercaptopropionic (4) and mercaptooctanoic (5) acid, were investigated by cyclic voltammetric, contact angle, FT-IR, and quartz crystal microbalance measurements in neutral aqueous media. The level of organization of the aggregates formed by the three CD derivatives (1−3) on the negatively charged surface of the mercaptopropionic and mercaptooctanoic acid modified gold electrodes proved to be substantially better than that on bare gold electrodes. This finding suggests that although the amphiphilic character of compounds 1−3 can induce aggregation on a gold surface, the electrostatic interaction between the carboxylic acid groups and the positively charged cyclodextrins is the primary force leading to the formation of well-organized aggregates.

DOI:10.1021/la970749w

1997

Formation of a new class of layered, microcrystalline polymers from a simple hydrolytic polycondensation of n-alkyltrichlorosilanes in water is demonstrated. The structure of the polymeric condensate, determined from a combination of spectroscopic, diffraction, and thermal analysis techniques, consists of highly uniform, pillared microcrystallites in which the inorganic siloxy backbones are present in periodic layers, each containing a monomolecular layer of intercalated water, separated by crystalline assemblies of alkyl chains. The alkyl-chain organization shows a remarkable resemblance to that in highly organized, self-assembled monolayers formed from the precursor silane molecules on hydrophilic substrates and this parallel lends support to the critical importance of water in monolayer self-assembly of silanes.

DOI:10.1021/ja963284p

Infrared reflection–absorption spectroscopy has been used to characterize thin overlayers (1–200 Å) of D2O ice deposited in UHV onto a set of self-assembled alkanethiolate monolayers(SAMs) of controlled wettabilities on gold. The SAMs were prepared from a series of controlled composition, mixed solutions of HS(CH2)15CH3 and HS(CH2)16OH, making it possible to investigate the whole wettability range from θ≈0° to θ=112°, where θ is the static contact angle with water. Dosing of D2O and infrared measurements were carried out at selected sample temperatures between 82 and 150 K. Experimental spectra of ice overlayers recorded below 100 K on all SAM substrates are in good agreement with simulated reflection–absorption spectra, derived from the optical constants of amorphous ice. This agreement allows accurate film thickness determination. In contrast, lack of correspondence in spectral signature is noted between the spectra of annealed films and simulated polycrystalline (or amorphous) icespectra. We interpret this discrepancy to suggest that significant substrate-induced differences between thin overlayers and bulk ice persist in the latter case. Spectral indications of ice–substrate interaction are also seen for amorphous ice, and are especially prominent in the case of highly hydrophobic (pure CH3-terminated, θ=112°) substrates. In this case the substrate effect extends up to an average film thickness (150–200 Å) corresponding to ∼50 icemonolayers, in contrast to highly hydrophilic OH-terminated substrates where the substrate effects appear to vanish beyond ∼5 monolayers (15–20 Å average thickness). Annealing of thin ice overlayers (2–3 monolayers) clearly demonstrates a strong correlation between the onset as well as progression of the transition from amorphous to polycrystallineice and the exact substrate wettability or chemical composition. The data further suggest the existence of metastable intermediate forms, that are neither purely amorphous nor polycrystalline. We discuss these observations in terms of substrate–overlayer interaction. A tentative “phase diagram” summarizing these results is presented.

DOI:10.1063/1.473049

Several patterned monolayers of alkanethiols CH3(CH2)n-1SH on a polycrystalline Au substrate were prepared by using microcontact printing and solution deposition methods, and their surfaces were examined by IR spectroscopy, scanning force microscopy, lateral force microscopy (LFM), and force modulation microscopy (FMM). Our work shows that LFM and FMM can detect differences in packing density of chemically identical molecules which are too small to be detected by IR, ellipsometry, and wetting measurements and suggests that the tip−sample contact area is an important parameter governing the contrasts of LFM and FMM images. Stiffness images obtained with FMM depend on changes in the Young's modulus of a sample surface as well as in the tip−sample contact area. As a result, a surface region of small modulus can have a large stiffness due to its large contact area.

DOI:10.1021/la960935m

1996

Mixed composition monolayers of similar n-alkanethiols on are formed by self-assembly. While the average surface composition of these films accurately reflects the composition of the deposition solution, scanning tunneling microscopy and secondary ion mass spectroscopy measurements show that the films phase separate on the nanometer scale. Scanning tunneling microscopy has been used to follow molecular motions within these films. We discuss our observations in terms of the formation and stability of the phase-segregated domains, and their potential importance in nanoscale applications.

DOI:10.1088/0957-4484/7/4/025

Highly organized monolayers formed from the self-assembly of octadecyl derivatives on oxide-covered Si and Ti substrates have been exposed to electron beam impact under typical conditions used in lithographic patterning. A combination of X-ray photoelectron spectroscopy, ellipsometry, infrared spectroscopy, and liquid drop contact angle measurements show that the major effect of irradiation is the loss of H, via cleavage of C−H bonds, to form a carbonaceous residue with a surface containing oxygenated functional groups.

DOI:10.1021/jp960705g

Dots demonstrating critical resist dimensions of approximately 5 to 6 nm were formed in an octadecylsiloxane monolayer on silicon by electron beam exposure using a digital scanning electron microscope at 20 keV beam energy. The patterned dots were observed by imaging with an atomic force microscope(AFM). The electron beam size was measured to confirm that it is not the limiting factor in the exposure resolution. The limit that prevents the observation of smaller structures is either the small contrast in the AFM imaging for smaller dots or an intrinsic material limit caused by the secondary electron range.

DOI:10.1063/1.115680

Self‐assembled monolayers of octadecylthiol on GaAs and octadecylsiloxanes on titanium,aluminum, and silicon have been used as electron beam resists for plasma etching into the substrates. An electron cyclotron resonance source was used to excite a low‐pressure, high‐density plasma with low ion energy to achieve high selectivity with the thin masking layers. Patterning of monolayers on GaAs usually produced a negative tone, but etching of the metal surfaces resulted in positive tone patterning. The maximum etch depth into the GaAs was ∼100 nm using the negative tone process. Lines have been etched into Ti with linewidths down to ∼20 nm. The negative tone process can be explained by the cross linking of the monolayer under high‐dose electron beam exposure; however, the positive tone process must rely on contrast either from different etching characteristics of the oxides or different structural arrangements of the different SAMs.

DOI:10.1116/1.580347

1995

This paper describes studies of the formation of self-assembled monolayers (SAMs) and multilayers on gold surfaces of rigid-rod conjugated oligomers that have thiol, alpha,omega-dithiol, thioacetyl, or alpha,omega-dithioacetyl end groups. The SAMs were analyzed using ellipsometry, X-ray photoelectron spectroscopy (XPS), and infrared external reflectance spectroscopy. The thiol moieties usually dominate adsorption on the gold sites; interactions with the conjugated pi-systems are weaker. Rigid rod alpha,omega-dithiols form assemblies in which one thiol group binds to the surface while the second thiol moiety projects upward at the exposed surface of the SAM. In situ deprotection of the thiol moieties by deacylation of thioacetyl groups using NH4OH permits formation of SAMs without having to isolate the oxidatively unstable free thiols. Moreover, direct adsorption, without exogenous base, of the thioacetyl-terminated oligomers can be accomplished to generate gold surface-bound thiolates. However, in the non-base-promoted adsorptions, higher concentrations of the thioacetyl groups, relative to that of thiol groups, are required to achieve monolayer coverage in a given interval. A,thiol-terminated phenylene-ethynylene system was shown to have a tilt angle of the long molecular axis of less than 20 degrees from the normal to the substrate surface. These aromatic alpha,omega-dithiol-derived monolayers provide the basis for studies leading to the design of molecular wires capable of bridging proximate gold surfaces.

DOI:10.1021/ja00142a021

Monolayers of n-octadecylsiloxane (CH3(CH2)(17)SiOxHy;ODS) were self-assembled from n-octadecyltrichlorosilane solutions onto a series of OH- and CH3-containing surfaces prepared from the self-assembly of controlled composition mixtures of HO(CH2)(16)SH and H3C(CH2)(15)SH on gold (RS/Au). Using null ellipsometry, infrared spectroscopy, and hexadecane contact angles; the coverages, chain structures, and surface wetting of the formed ODS assemblies were determined as a function of the OH fraction, f(OH) = [OH]/[CH3 + OH], in the starting RS assembly. Three distinct ODS adsorption regimes were observed: (1) on pure CH3 surfaces no stable adsorbed layer forms; (2) for 0.1 less than or similar to f(OH) less than or similar to 0.8, the coverage is incomplete and monotonically increases with f(OH) and the ODS structures consist of a range of coexisting domains of nearly all-trans chains and disordered, liquid-like components with maximum disorder content, estimated as > 80%, arising near f(OH) similar to 0.5; and (3) for f(OH) > 0.8, a high coverage, close-packed monolayer is formed with predominantly all-trans chains tilted at 8-12 degrees from the surface normal, a distinctly different tilt than the known value of 26-30 degrees for the RS underlayer and an indication of strong structural decoupling (incommensurability) between the two highly organized layers. The f(OH)-dependence of the structures is explained on the basis of a previously proposed hypothesis that a continuous preadsorbed, substrate-bound water film is required for achieving maximum organization during n-alkylsiloxane self-assembly and that, in the present case of the OH/CH3 surfaces, the required water film structure at the preparation solution/substrate interface is not reached until high f(OH) values.

DOI:10.1021/j100024a049

Monolayers of n-octadecylsiloxane (ODS) have been prepared by Langmuir-Blodgett film transfer of pre-polymerized films onto oxidized silicon substrates at a surface pressure of 20 mN.m(2). Characterization by infrared spectroscopy, single-wavelength ellipsometry, and contact angle measurements show that highly organized structures are formed which are nearly identical to those reported for ODS films made by solution self-assembly. Atomic force microscopy images reveal that the macroscopic structure of the LB film consists of large, dense domains on the size scale of similar to 10 mu m.

DOI: 10.1021/bk-1995-0615.ch022

Self‐assembled monolayers of octadecylsiloxane and octadecylthiol have been modified by high‐resolution electron beam lithography. Focused electron beams from 1 to 50 keV and scanning tunneling microscopy at ∼10 eV have been used as patterning tools. The patterns have been transferred into many substrates by wet, dry, and combinations of wet and dry etches. Wet etching almost always results in a positive tone, but reactive ion etching of GaAs with Cl2 at very low dc biases ( less than 10 V) results in a negative tone. The effect of electron beam damage on the monolayers and the subsequent etching reactions has been explored through x‐ray photoelectron spectroscopy.

DOI:10.1116/1.588225

We address the longstanding issue of substrate effects in alkylsiloxane monolayer self-assembly. With proper substrate prehydration, we observe formation of identical quality, compact, quasi-crystalline monolayers on oxidized silicon and gold substrates with widely different chemical character, the former capable of covalent bonding to the adsorbed molecules via silanol groups and the latter devoid of reactive surface sites. Infrared spectroscopy, ellipsometry, and wetting measurements show identical average film structures consisting of highly extended chains tilted at 10 (+/-2)degrees with significant end-gauche defect content. This observed substrate independence is consistent with our previous hypothesis that substrate-bound water promotes the decoupling of the organic film from the underlying solid surface.

DOI:10.1021/la00007a007

1994

Scanning tunneling microscopy was used to probe the molecular scale structures of pure and mixed composition self‐assembled monolayers of CH3(CH2)15SH and CH3O2C(CH2)15SH on gold. A predominant defect structure is found for both films, and is assigned as a void defect in the thiolate overlayer. The mixed composition films are observed to phase segregate into domains of the pure component thiolates. Time‐lapse series of scanning tunneling microscope images reveal kink‐driven motion at step edges and exchange of thiolate molecules on terraces.

DOI:10.1116/1.587689

Scanning tunneling microscopy has been used to demonstrate phase segregation in varied composition, two-component self-assembled monolayers on gold. These monolayer films were assembled using CH3(CH2)(15)SH and CH3O2C(CH2)(15)SH, two similar alkanethiol molecules which are non-hydrogen-bonding and have identical alkyl chain lengths. X-ray photoelectron spectroscopy, infrared spectroscopy, and ellipsometry have been used to characterize the average chemical compositions and average molecular structures of these films. Scanning tunneling microscopy of single composition, self-assembled monolayers of each of these molecules shows a preponderance of defects which can be attributed to single missing chains. In mixed composition films, we observe nanometer scale molecular domains with time-dependent shapes. These observations have important implications both for the fundamental understanding of solubilities and phase segregation in quasi-two-dimensional mixtures and for applications of self-assembly in which spatial patterns of adsorbate mixtures are important.

10.1021/j100082a040

We propose a new mechanism for diffusion of surface adsorbates in which motion of the substrate atoms to which the adsorbates are attached results in the motion of the substrate-adsorbate complex. We show an experimental example-the motion of self-assembled monolayers of CH3O2C(CH2)(15)SH on gold which we have observed by time-lapse imaging using scanning tunneling microscopy. This mechanism is also used to explain previous data for motion of Cu-O complexes on the Cu{110} surface.

DOI:10.1021/j100094a024

We have studied the effect of preparation temperature, in the range 5-65 degrees C, on the structures of n-octadecylsiloxane monolayers prepared by self-assembly from dilute solution of n-octadecyltrichlorosilane onto the surface of freshly hydrated, oxidized silicon substrates. Structural features of the films were characterized using a combination of liquid drop contact angle measurements, null ellipsometry, and infrared transmission spectroscopy. The contact angle data confirm a previously reported observation of a critical temperature, T-c similar to 28 +/- 5 degrees C, below which the surface energy is constant at a near-limiting value of a pure CH3 surface and above which the surface energy monotonically increases with increasing temperature. Coverages and chain organization, as measured by ellipsometry and vibrational spectroscopic features (peak positions and integrated intensities of methylene C-H stretching modes), respectively, show changes in the same temperature region as the wetting behavior. We conclude that when prepared below T-c, the films exhibit a heterogeneous structure with closely spaced islands of densely packed, nearly all-trans alkyl chains arranged nearly vertical to the surface. In contrast, when prepared above T-c, the films exhibit monotonically diminishing coverage with increasing preparation temperature and the alkyl chains increasingly assume higher contents of conformational disorder. Further, the infrared data indicate that these higher temperature films are heterogeneous with coexisting domains of high and low chain conformational ordering. All the data, taken together, are in good conformity with a film formation mechanism which involves, prior to siloxy group cross-linking, the intervention of intermediate structural phases of mobile alkylsiloxy species adsorbed on a water layer adjacent to the solid substrate surface. In support of this mechanism, a strong parallel is apparent between T-c and the triple point temperature at which gas (G), liquid-expanded (LE), and liquid-condensed (LC) phases coexist for C-18 chain Langmuir monolayers at the air/bulk water interface. Below T,the self-assembled film structure is similar to that of the nearly pure LC Langmuir phase while above T-c the film structure is similar to that of coexisting LE and LC phases. Deviations of the self-assembled film structures for the analogous equilibrium Langmuir phase structures occur at higher preparation temperatures and are rationalized in terms of both the known occurrence of nonequilibrium phases in Langmuir films above the triple point temperature and the relative acceleration of the Si-O-Si cross-linking reaction in the self-assembled film to form structures with frozen-in defects.

DOI:10.1021/j100082a031

Self‐assembled monolayers have been modified with focused electron beams of energy 1–50 keV and scanning tunneling microscopy(STM) based lithography with energies of ∼10 eV. Modifications ∼15 nm in size have been formed by STM and ∼25 nm in size by 50 keV beams. The fact that these materials work as self‐developing electron beam resists is demonstrated by both atomic force microscopy imaging and pattern transfer using conventional wet etchants. Patterns have been transferred to silicon substrates to a depth of ≳120 nm with a multistep wet etching process. The mechanism of electron beam modification has also been explored to better design future monolayer processes.

DOI:10.1116/1.587635

Partial monolayers of uniform length alkyl chains [CH3(CH2) n –; n=10–21] bonded at constant coverage onto a disordered lattice of silanol groups on amorphous SiO2, exhibit a distinct change in surfacewetting behavior for 14≤n≤17 which correlates closely with a significant shift in the average conformational ordering of the chains. These data provide definitive evidence for the existence of a family of surface‐constrained, intermediately ordered phases of discrete length, flexible chains and imply that broad classes of these families exist.

DOI:10.1063/1.466604

1993

We demonstrate the spectroscopic capabilities of an ac scanning tunneling microscope. This ac scanning tunneling microscope has a bias voltage modulation frequency tunable over the range dc–20 GHz and is thus capable of recording images and local spectra of insulating as well as conducting substrates. In this article we detail the spectral sensitivity and the tip‐sample separation dependence of the spectra recorded by simultaneously tuning the modulation and detection frequencies. Spectra of an evaporated goldfilm and of self‐assembled monolayers of CH3(CH2)15SH and of CH3O2C(CH2)15SH on gold are presented.

DOI: 10.1116/1.578339

It was demonstrated that self‐assembled monolayers of n‐octadecanethiol [ODT; CH3(CH2)17SH] on GaAs and n‐octadecyltrichlorosilane [OTS; CH3(CH2)17SiCl3] on SiO2 act as self‐developing positive electron beam resists with electron‐beam sensitivities of ∼100–200 μC/cm2. For the OTS monolayer on a silicon native oxide, atomic force microscopy (AFM) images of the exposed layer before etching demonstrate the removal of all or part of the layer upon electron‐beam exposure. Features as small as 25 nm were resolvable in a 50 nm period grating. A resist contrast curve for OTS was obtained from AFM depth measurements as a function of dose. An ammonium hydroxide water etch was used to transfer patterns into the GaAs to a depth of at least 30 nm and buffered HF was used for pattern transfer into the SiO2 to a depth of at least 50 nm.

DOI: 10.1116/1.586609

1992

We now report the discovery of a new class of organized monolayers derived from the self-assembly of alkanethiols directly onto the bare GaAs (100) surface. That viable S/GaAs chemistry might exist to allow self-assembly has been indicated by current studies which show that room temperature coverage of GaAs by deposits of inorganic sulfide salts,' P2S5,12a nd simple organothiol compounds (six carbons or less) exerts significant effects on electron-hole pair recombination velocities. We have produced SAMs from alkanethiols, X(CH,),SH for X = CH3 with n = 11-21 and X = C02H and C02CH3 with n = 15. The bulk of our characterization has been on octadecanethiol (ODT, X = CH,, and n = 17), and for brevity we focus on these results which show that the monolayer consists of a stable, highly organized assembly of tilted, conformationally ordered alkyl chains, chemically bonded directly to the bare GaAs surface.

DOI: 10.1021/ja00030a076

A semitheoretical formalism based on classical electromagnetic wave theory has been developed for application to the quantitative treatment of reflection spectra from multilayered anisotropicfilms on both metallic and nonmetallic substrates. Both internal and external reflection experiments as well as transmission can be handled. The theory is valid for all wavelengths and is appropriate, therefore, for such experiments as x‐ray reflectivity, uv–visible spectroscopic ellipsometry, and infrared reflection spectroscopy. Further, the theory is applicable to multilayered filmstructures of variable number of layers, each with any degree of anisotropy up to and including full biaxial symmetry. The reflectivities (and transmissivities) are obtained at each frequency by solving the wave propagation equations using a rigorous 4×4 transfer matrix method developed by Yeh in which the optical functions of each medium are described in the form of second rank (3×3) tensors. In order to obtain optical tensors for materials not readily available in single crystal form, a method has been developed to evaluate tensor elements from the complex scalar optical functions (n̂) obtained from the isotropic material with the limitations that the molecular excitations are well characterized and obey photon–dipole selection rules. This method is intended primarily for infrared vibrational spectroscopy and involves quantitative decomposition of the isotropic imaginary optical function (k) spectrum into a sum of contributions from fundamental modes, the assignment of a direction in molecular coordinates to the transition dipole matrix elements for each mode, the appropriate scaling of each k vector component in surface coordinates according to a selected surface orientation of the molecule to give a diagonal im(n̂) tensor, and the calculation of the real(n̂) spectrumtensor elements by the Kramers–Kronig transformation. Tensors for other surface orientations are generated by an appropriate rotation matrix operation. To test the viability of this approach, three sets of experimentally derived infrared spectra of oriented monolayer assemblies on quite distinctively different substrates were chosen for simulation: (1) n‐alkanethiols self‐ assembled onto gold, (2) n‐alkanoic acid salt Langmuir–Blodgett (LB) monolayers on carbon, and (3) n‐alkanoic acid salt LB monolayers on silica glass. The formalism developed was used to simulate the spectral response and to derive structural features of the monolayers. Good agreement was found where comparisons with independent studies could be made and, in general, the method appears quite useful for structural studies of highly organized thin films.

DOI: 10.1063/1.462847

1991

Long-chain alkanethiols, HS(CH2),CH3, adsorb from solution onto the surfaces of gold, silver, and copper and form monolayers. Reflection infrared spectroscopy indicates that monolayers on silver and on copper (when carefully prepared) have the chains in well-defined molecular orientations and in crystalline-like phase states, as has been observed on gold. Monolayers on silver are structurally related to those formed by adsorption on gold, but different in details of orientation. The monolayers formed on copper are structurally more complex and show a pronounced sensitivity to the details of the sample preparation. Quantitative analysis of the IR data using numerical simulations based on an average single chain model suggests that the alkyl chains in monolayers on silver are all-trans zig-zag and canted by - 12 degree from the normal to the surface. The analysis also suggests a twist of the plane containing the carbon backbone of -45 degree from the plane defined by the tilt and surface normal vectors. For comparison, the monolayers that form on adsorption of alkanethiols on gold surfaces, as judged by their vibrational spectra, are also trans zig-zag extended but, when interpreted in the context of the same single chain model, have a cant angle of - 2 7 O and a twist of the plane of the carbon backbone of -53 degree. The monolayers formed on copper (when they are obtained in high quality) exhibit infrared spectra effectively indistinguishable from those on silver and thus appear to have the same structure. Films on copper are also commonly obtained that are structurally ill-defined and appear to contain significant densities of gauche conformations. These spectroscopically based interpretations are compatible with inferences from wetting and XPS measurements. The structure of the substrate-sulfur interface appears to control molecular orientations of the alkyl groups in these films. An improved structural model, incorporating a two-chain unit cell and allowing for the temperature-dependent population of gauche conformations, is presented and applied to the specific case of the structures formed on gold.

DOI: 10.1021/ja00019a011

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