Accurate modeling of the cellular microenvironment is important for improving studies of cell biology in vitro. Here, we demonstrate a flexible method for creating a cellular microenvironment in vitro that allows i) controlled spatial distribution (patterning) of multiple types of cells within three-dimensional (3-D) matrices of a biologically-derived, thermally-curable hydrogel (Matrigel) and ii) application of gradients of soluble factors, such as cytokines, across the hydrogel. The technique uses laminar flow to divide a microchannel into multiple subchannels separated by microslabs of hydrogel. It does not require the use of UV light or photoinitiators, and is compatible with cell culture in the hydrogel. This technique makes it possible to design model systems to study cellular communication mediated by the diffusion of soluble factors within 3-D matrices. Such factors can originate either from secretions of neighboring cells patterned within the microchannel, or from an external source-e.g., a solution of growth factors injected into a subchannel. This method is particularly useful for studying cells such as those of the immune system, which are often weakly adherent and difficult to position precisely with standard systems for cell culture. We demonstrated this application by co-culturing two types of macrophage-like cells (BAC1.2F5 and LADMAC cell lines) within spatially separated regions of a slab of hydrogel. This pair of cell lines represents a simple model system for intercellular communication: the LADMAC cells produce colonystimulating factor 1 (CSF-1), which is required by the BAC cells for survival.

This paper describes the fabrication of a fluidic device for detecting and separating diamagnetic materials that differ in density. The basis for the separation is the balance of the magnetic and gravitational forces on diamagnetic materials suspended in a paramagnetic medium. The paper demonstrates two applications of separations involving particles suspended in static fluids for detecting the following: (i) the binding of streptavidin to solid-supported biotin and (ii) the binding of citrate-capped gold nanoparticles to amine-modified polystyrene spheres. The paper also demonstrates a microfluidic device in which polystyrene particles that differ in their content of CH2Cl groups are continuously separated and collected in a flowing stream of an aqueous solution of GdCl3. The procedures for separation and detection described in this paper require only gadolinium salts, two NdFeB magnets, and simple microfluidic devices fabricated from poly(dimethylsiloxane). This device requires no power, has no moving parts, and may be suitable for use in resource-poor environments.

Cost-effective and convenient methods for fabrication of patterned metallic nanostructures over the large (mm2) areas required for applications in photonics are much needed. In this paper, we demonstrate the fabrication of arrays of closed and open, loop-shaped nanostructures by a technique (nanoskiving) that combines thin-film deposition by metal evaporation with thin-film sectioning. These arrays of metallic structures serve as frequency-selective surfaces at mid-infrared wavelengths. Experiments with structures prepared using this technique demonstrate that a closed-looped structure has a single dominant resonance regardless of the polarization of the incident light, while open structures have resonances that are anisotropic with respect to the polarization of the electric field. Finite-difference time-domain (FDTD) simulations reproduce the scattering spectra of these frequency-selective surfaces, provide an explanation of the wavelength of the experimentally observed resonances, and rationalize their polarization dependence based on the patterns of current induced in the nanostructures.

This paper describes a synthetic dimer of carbonic anhydrase, and a series of bivalent sulfonamide ligands with different lengths (25 to 69 Å between the ends of the fully extended ligands), as a model system to use in examining the binding of bivalent antibodies to antigens. Assays based on analytical ultracentrifugation and fluorescence binding indicate that this system forms cyclic, noncovalent complexes with a stoichiometry of one bivalent ligand to one dimer. This dimer binds the series of bivalent ligands with low picomolar avidities (Kdavidity = 3 – 40 pM). A structurally analogous monovalent ligand binds to one active site of the dimer with Kdmono = 16 nM. The bivalent association is thus significantly stronger (Kdmono/Kdavidity ranging from ~500 to 5000 unitless) than the monovalent association. We infer from these results, and by comparison of these results to previous studies, that bivalency in antibodies can lead to associations much tighter than monovalent associations (although the observed bivalent association is much weaker than predicted from the simplest level of theory—predicted Kdavidity of ~ 0.002 pM and Kdmono/Kdavidity ~ 8 × 106 unitless).

Ca2+-triggered neurotransmitter release depends on the formation of SNARE complexes that bring the synaptic vesicle and plasma membranes together, on the Ca2+ sensor synaptotagmin-1, and on complexins, which play active and inhibitory roles. Release of the complexin inhibitory activity by binding of synaptotagmin-1 to the SNARE complex, causing complexin displacement, was proposed to trigger exocytosis. However, the validity of this model was questioned based on the observation of simultaneous binding of complexin-I and a fragment containing the synaptotagmin-1 C2 domains (C2AB) to membrane-anchored SNARE complex. Using diverse biophysical techniques, here we show that C2AB and complexin-I do not bind to each other but can indeed bind simultaneously to the SNARE complex in solution. Hence, the SNARE complex contains separate binding sites for both proteins. However, total internal reflection fluorescence microscopy experiments show that C2AB can displace a complexin-I fragment containing its central SNARE-binding helix and an inhibitory helix (Cpx26–83) from membrane-anchored SNARE complex under equilibrium conditions. Interestingly, full-length complexin-I binds more tightly to membrane-anchored SNARE complex than Cpx26–83, and is not displaced by C2AB. These results show that interactions of N- and/or C-terminal sequences of complexin-I with the SNARE complex and/or phospholipids increase the affinity of complexin-I for the SNARE complex, hindering dissociation induced by C2AB. We propose a model whereby binding of synaptotagmin-1 to the SNARE complex directly or indirectly causes a rearrangement of the complexin-I inhibitory helix without inducing complexin-I dissociation, thus relieving the inhibitory activity and enabling cooperation between synaptotagmin-1 and complexin-I in triggering release.