Lori A. Lepak

An endothelial and astrocyte co-culture model of the blood–brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane

The endothelial cells comprising brain capillaries have extremely tight intercellular junctions which form an essentially impermeable barrier to passive transport of water soluble molecules between the blood and brain. Several in vitro models of the blood-brain barrier (BBB) have been studied, most utilizing commercially available polymer membranes affixed to plastic inserts. There is mounting evidence that direct contact between endothelial cells and astrocytes, another cell type found to have intimate interaction with the brain side of BBB capillaries, is at least partially responsible for the development of the tight intercellular junctions between BBB endothelial cells. However, the membranes commonly used for BBB in vitro models are lacking certain attributes that would permit a high degree of direct contact between astrocytes and endothelial cells cultured on opposing sides. This work is based on the hypothesis that co-culturing endothelial and astrocyte cells on opposite sides of an ultra-thin, highly porous membrane will allow for increased direct interaction between the two cell types and therefore result in a better model of the BBB. We used standard nanofabrication techniques to make membranes from low-stress silicon nitride that are at least an order of magnitude thinner and at least two times more porous than commercial membrane inserts. An experimental survey of pore sizes for the silicon nitride membranes suggested pores approximately 400 nm in diameter are adequate for restricting astrocyte cell bodies to the seeded side while allowing astrocyte processes to pass through the pores and interact with endothelial cells on the opposite side. The inclusion of a spun-on, cross-linked collagen membrane allowed for astrocyte attachment and culture on the membranes for over two weeks. Astrocytes and endothelial cells displayed markers specific to their cell types when grown on the silicon nitride membranes. The transendothelial electrical resistances, a measure of barrier tightness, of endothelial and astrocyte co-cultures on the silicon nitride membranes were comparable to the commercial membranes, but neither system showed synergy between the two cell types in forming a tighter barrier. This lack of synergy may have been due to the loss of ability of commercially available primary bovine brain microvascular endothelial cells to respond to astrocyte differentiating signals.

The site-specific reactivity of isopropanol in aqueous silicon etching: Controlling morphology with surface chemistry

The site-specific rates of isopropanol reaction on Si(111) were studied by kinetic competition with an etchant of known anisotropy and quantified using concentration-dependent changes in the etched surface morphology using a combination of STM measurements and kinetic Monte Carlo simulations. The isopropoxide ion was identified as the active species.

Direct Casting of Polymer Membranes into Microfluidic Devices

ABSTRACT Fully functional lab-on-a-chip devices for biological analyses require the capability for cell culture, separation, and purification, as well as analyses to be integrated on a single platform. To date, a great deal of research has been focused on analytical methods for the miniaturization of column-based separations. We have created a platform that provides the capability of including membrane separations as an intermediate stage in such devices. Our techniques adapt conventional silicon processing methods to the casting of membranes directly onto the silicon substrate through a wet inversion process. This process allows precise control of membrane thickness and pore size distribution based on the processing conditions. Using this methodology, we were able to fabricate devices that were found to be very robust with molecular weight cutoffs of approximately 350 Da as measured by solute flux in a dialysis mode of operation. These devices were also found to be suitable for cell culture, as evidenced by the high viability of fibroblasts grown within our device. On the basis of these results, a wide range of separations and coculture applications are possible.

Improvement of carrier mobility of top-gated SiC epitaxial graphene transistors using a PVA dielectric buffer layer

The effects of treatment with polyvinyl alcohol (PVA) and a dielectric film of HfO(2) on the properties of SiC based epitaxial graphene have been explored and analyzed. We have characterized the carrier mobility of graphene on Si-face and C-face SiC with a layer of HfO(2), with or without an initial PVA treatment on the device active layer. Epitaxial graphene grown on the C-face displays a higher mobility than a film grown on the silicon face. Also, the mobility in the presence of the PVA treatment with HfO(2) dielectric layer has been improved, compared with the mobility after deposition of only gate dielectric: ∼20% in C-face graphene and ∼90% in Si-face graphene. This is a major improvement over the degradation normally observed with dielectric/graphene systems.

Separation Characteristics of On‐chip Biopolymer Membranes

Abstract Research in miniaturization of microfluidic devices and total analysis systems for chemical and biological applications has been active for more than a decade. Sample size and processing time have decreased dramatically, while sensitivity has increased, along with the ability to run multiple tests in parallel. However, sensitivity depends in most cases on the purification or enrichment of the sample prior to loading onto a device. Filtration is performed either prior to sample loading or through membranes adhered to the substrate. Currently, these membranes cannot be patterned to micrometer resolution and the adhesion process may be incompatible with the fabrication and/or may introduce contaminants to the process. We have developed a process to incorporate filtration onto microfluidic devices that is compatible with microfabrication methods and is suitable for biological applications. We cast cellulose acetate membranes directly onto silicon wafers without the use of adhesives and studied the filtration properties of these membranes using compounds with molecular weights ranging from 100 to 900. We also have varied the casting conditions and studied the effects of these variations on the membranes rejection characteristics. Cellulose acetate membranes, cast directly onto silicon wafers, adhered well to the substrate and had high structural integrity. We have developed membranes with four different molecular weight cut‐offs; 300, 350, 600, and 700Da. Parameters such as solubility and charge were also investigated for their contribution to the rejection characteristics.

Preparation and Characterization of Porous Collagen Membranes on Silicon

Advances in biotechnology in the past decade have raised the possibility of fabricating biocompatible, porous membranes for molecular sieving and dialysis separations of particles sized 20–50 nm or less. As a prerequisite for such applications, we demonstrate that thin films (∼ 400 nm) of monomeric bovine dermal collagen spin-deposited on a silicon substrate are patternable using standard semiconductor microlithographic processing techniques. Patterning via liftoff has reliably produced square features as small as 10–25 μm laterally, and 50 nm thick, in initial experiments. HVEM (high vacuum electron microscope) images of these collagen membranes have revealed typical pore sizes ranging from 1–100 nm. Through-membrane diffusion of chromophores spanning this size range was quantified via UV/vis spectrometry. These studies revealed that a 400 nm thick collagen membrane crosslinked with 0.02% glutaraldehyde rejected detectable quantities of methyl orange dye (MW 327) for at least 48 hours, while a 100 nm thick layer admitted methyl orange in under 30 minutes. DNA has been demonstrated to pass through a 100 nm thick collagen layer more slowly than through a bare through-etched control wafer.