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The fabrication of nanostructured films possessing tricontinuous minimal surface mesophases with well-defined framework and pore connectivity remains a difficult task. As a new route to these structures, we introduce glycerol monooleate (GMO) as a template for evaporation-induced self-assembly. As deposited, a nanostructured double gyroid phase is formed, as indicated by analysis of grazing-incidence small-angle x-ray scattering data. Removal of GMO by UV/O(3) treatment or acid extraction induces a phase change to a nanoporous body-centered structure which we tentatively identify as based on the IW-P surface. To improve film quality, we add a co-surfactant to the GMO in a mass ratio of 1:10; when this co-surfactant is cetyltrimethylammonium bromide, we find an unusually large pore size (8-12 nm) in acid extracted films, while UV/O(3) treated films yield pores of only ca. 4 nm. Using this pore size dependence on film processing procedure, we create a simple method for patterning pore size in nanoporous films, demonstrating spatially-defined size-selective molecular adsorption.

Background The desire to immobilize, encapsulate, or entrap viable cells for use in a variety of applications has been explored for decades. Traditionally, the approach is to immobilize cells to utilize a specific functionality of the cell in the system. Scope of review This review describes our recent discovery that living cells can organize extended nanostructures and nano-objects to create a highly biocompatible nano//bio interface [1]. Major conclusions We find that short chain phospholipids direct the formation of thin film silica mesophases during evaporation-induced self-assembly (EISA) [2], and that the introduction of cells alter the self-assembly pathway. Cells organize an ordered lipid-membrane that forms a coherent interface with the silica mesophase that is unique in that it withstands drying—yet it maintains accessibility to molecules introduced into the 3D silica host. Cell viability is preserved in the absence of buffer, making these constructs useful as standalone cell-based sensors. In response to hyperosmotic stress, the cells release water, creating a pH gradient which is maintained within the nanostructured host and serves to localize lipids, proteins, plasmids, lipidized nanocrystals, and other components at the cellular surface. This active organization of the bio/nano interface can be accomplished during ink-jet printing or selective wetting–processes allowing patterning of cellular arrays–and even spatially-defined genetic modification. General significance Recent advances in the understanding of nanotechnology and cell biology encourage the pursuit of more complex endeavors where the dynamic interactions of the cell and host material act symbiotically to obtain new, useful functions. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

The nanostructure of silica and hybrid thin film mesophases templated by phospholipids via an evaporation-induced self-assembly (EISA) process was investigated by grazing-incidence small-angle X-ray scattering (GISAXS). Diacyl phosphatidylcholines with two tails of 6 or 8 carbons were found to template 2D hexagonal mesophases, with the removal of lipid from these lipid/silica films by thermal or UV/O3 processing resulting in a complete collapse of the pore volume. Monoacyl phosphatidylcholines with single tails of 10–14 carbons formed 3D micellular mesophases; the lipid was found to be extractable from these 3D materials, yielding a porous material. In contrast to pure lipid/silica thin film mesophases, films formed from the hybrid bridged silsesquioxane precursor bis(triethoxysilyl)ethane exhibited greater stability toward (both diacyl and monoacyl) lipid removal. Ellipsometric, FTIR, and NMR studies show that the presence of phospholipid suppresses siloxane network formation, while actually promoting condensation reactions in the hybrid material. 1D X-ray scattering and FTIR data were found to be consistent with strong interactions between lipid headgroups and the silica framework.

When lipid-directed assembly of silicic acid precursors is conducted in the presence of living cells, the cells intervene, surrounding themselves with a fluid, multilayered lipid vesicle that interfaces coherently with an ordered silica mesophase. This bio/nano interface is unique in that its uniform nanostructure prevents excessive drying of water, maintaining cell viability, yet provides accessibility of the cell surface to small molecules. In comparison to existing immobilization schemes, such as encapsulation within sol-gel matrices, we show this interface to form by an active interplay between the living cell and surrounding matrix, which we refer to as cell-directed assembly (CDA). Importantly and perhaps uniquely, CDA creates a localized nanostructured microenvironment within which three-dimensional chemical gradients are established and maintained.

Amphiphilic phospholipids were used to direct the formation of biocompatible, uniform silica nanostructures in the presence of Saccharomyces cerevisiae and bacterial cell lines. The cell surfaces organize multilayered phospholipid vesicles that interface coherently with the silica host and help relieve drying stresses that develop with conventional templates. These host structures maintain cell accessibility, addressability, and viability in the absence of buffer or an external fluidic architecture. The cell surfaces are accessible and can be used to localize added proteins, plasmids, and nanocrystals. Prolonged cell viability combined with reporter protein expression enabled stand-alone cell-based sensing.

We present and evaluate a new approach to cell immobilization for use in cell-based biosensors. We have fabricated a microfluidic channel using poly(dimethylsiloxane) (PDMS) with cell entrapment posts for the gentle packing and immobilization of yeast cells. This method of immobilization allows for a density of metabolically active cells greater than 8.0 × 10 6 cells/mm 3 . The packed microcolumn approach addresses simple diffusional limitations inherent in traditional suspension and membrane entrapment techniques. By utilizing genetically engineered whole cells, rather then cellular components, the sensor is capable of detecting and responding to a wide range of biologically active compounds. In this study, Saccharomyces cerevisiae was genetically engineered to produce yellow fluorescent protein (YFP) when exposed to galactose. Fluorescence response of packed cells (G 1 phase) to galactose required 40% longer than the fluorescent response of cells grown in suspension. To address concerns of long-term viability (>60 days) and cell overgrowth, stationary phase cells were also tested in the microfluidic channel. Response time required approximately 50% longer than non-stationary phase cells packed inside the microfluidic channel. Additionally, cellular response as a function of the target analyte concentration was investigated and response time versus analyte concentration is reported. This report demonstrates proof-of-concept of using protein expression-based biosensors, based upon a packed, microcolumn architecture, as a dependable long-term storage platform.

Immobilization of individual cells and collections of cells in well-defined, reproducible, nano-to-microscale structures that allow structural and functional manipulation and interrogation is important for developing new classes of biotic/abiotic materials, for establishing the relationship between genotype and phenotype, and for elucidating responses to disease, injury/stress, or therapy - primary goals of biomedical research. Although there has been considerable recent progress in investigating the response of cells to chemical or topological patterns defined lithographically on 2D surfaces, it is time to advance from two-dimensional adhesion on dishes/fluidic devices to three-dimensional architectures that better represent the nanoporous, 3-D extracellular matrix (ECM). 3D immobilization in nanostructured hosts enables cells to be surrounded by other cells, maintains fluidic connectivity/accessibility, and allows development of 3-D molecular or chemical gradients that provide an instructive background to guide cellular behavior. Although 3-D cell immobilization in polymers, hydrogels, and inorganic gels has been practiced for decades, these approaches do not provide for bio/nano interfaces with 3D spatial control of topology and composition important to both the maintenance of natural cellular behavior patterns and the development of new non-native behaviors and functions. This LDRD project exploited our discovery of the ability of living cells to organize extended nanostructures and nano-objects in a manner that creates a unique, highly biocompatible bio/nano interface, mimicking the ECM, and maintaining cell viability, accessibility, and functionality (Baca et al. Science, 2006). Briefly, we found that, using short chain phospholipids to direct the formation of thin film silica mesophases during evaporation-induced self-assembly (EISA, Lu, Brinker et al. Nature 1997), the introduction of cells (yeast, Gram negative and positive bacteria, and several mammalian cells) alters profoundly the inorganic self-assembly pathway. Cells actively organize around themselves an ordered, multilayered lipid-membrane that interfaces coherently with a lipid-templated silica nanostructure. This bio/nano interface is unique in that it withstands drying (even evacuation) without cracking or the development of tensile stresses - yet it maintains accessibility to molecules, proteins/antibodies, plasmids, etc - introduced into the 3D silica host. Additionally cell viability is preserved for weeks to months in the absence of buffer or a fluidic architecture, making these constructs useful as standalone cell-based sensors. (On this basis, our sensors were launched to the space station for viability studies after exposure to vacuum and UV). The bio/nano interfaces we describe do not form 'passively' - rather they are a consequence of the cell's ability to sense and actively respond to external stimuli. During EISA, solvent evaporation concentrates the extracellular environment in osmolytes. In response to this hyperosmotic stress, the cells release water, creating a gradient in pH (and presumably other molecular components), which is maintained within the adjoining nanostructured host and serves to localize lipids, proteins, plasmids, lipidized nanocrystals, and a variety of other components at the cellular surface. This active organization of the bio/nano interface, which we refer to as cell directed assembly (CDA) can be accomplished during ink-jet printing or selective wetting - processes allowing patterning of cellular arrays - what's more we find that cells printed onto preformed, fluid lipid/silica mesophases integrate themselves within the silica nanostructure, creating a 3D environment essentially indistinguishable from that in CDA. We refer to this latterprocess as cell-directed integration (CDI). The synthetic constructs we have developed have allowed us to explore several fundamental questions concerning the mechanisms by which cells actively control nanostructure formation and function and conversely the mechanisms by which nanostructured interfaces, matrices, and patterns can control cellular behavior.

When lipid-directed assembly of silicic acid precursors is conducted in the presence of living cells, the cells intervene, surrounding themselves with a fluid, multilayered lipid vesicle that interfaces coherently with an ordered silica mesophase. This bio/nano interface is unique in that its uniform nanostructure prevents excessive drying of water, maintaining cell viability, yet provides accessibility of the cell surface to small molecules. In comparison to existing immobilization schemes, such as encapsulation within sol-gel matrices, we show this interface to form by an active interplay between the living cell and surrounding matrix, which we refer to as cell-directed assembly (CDA). Importantly and perhaps uniquely, CDA creates a localized nanostructured microenvironment within which three-dimensional chemical gradients are established and maintained.