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This paper describes the use of a layer-by-layer nanocoating technique for the encapsulation of insulin-producing pancreatic beta-cell spheroids (pseudoislets) within chitosan/alginate multilayers. We used pseudoislets self-organized from a population of the insulinoma cell line MIN6, derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta-cells, as an experimental model for the study of cell nanoencapsulation. The maintenance of spheroid morphology and retention of cell viability and metabolic functionality was demonstrated postencapsulation. By depositing an additional protein-repelling phosphorylcholine-modified chondroitin-4-sulfate layer, the coatings were found to shield effectively access of large molecules of the immune systems to the antigen-presenting cell surfaces. Transmission electron microscopy analysis of the encapsulated pseudoislets revealed that the coating did not damage the cell structure. In addition, nanoencapsulation permits the cells to respond to changes in extracellular glucose and other insulin secretagogues by releasing insulin with a profile similar to that of nonencapsulated cells. These results suggest that this nanofilm encapsulation technique has the characteristics required for the efficient transplantation of cellular engineered beta-cells as a cell replacement therapy for type 1 diabetes. This encapsulation method is general in scope and has implications for use in a variety of cellular therapeutics employing engineered tissues from cells generated in vitro from various sources, including those using genetic and cellular engineering techniques.

This paper describes a new and simple microarray platform for presenting multiple nonderivatized oligosaccharides to protein targets, with utility for mapping carbohydrate-protein recognition events. The approach is based on the creation of a hydrazide-derivatized, self-assembled monolayer on a gold surface in a single or two-step procedure, for efficient and selectively oriented anchoring of oligosaccharide probes via their reducing ends, with detection using fluorescence detection of bound proteins. The biggest hurdles in employing gold-based substrate for fluorescence-based microarray detection include fluorescence quenching and nonspecific surface adsorption of proteins. We found that the quenching effect could be minimized by introducing a omega-thiolated fatty acid (C16) self-assembled monolayer between the gold surface and hydrazide groups, followed by detection involving three successive binding protein layers covering the gold surface. In addition, an effective blocking scheme involving poly(ethylene glycol) aldehyde and bovine serum albumin was employed to reduce nonspecific protein adsorption to the chip surface. As proof of principle, we demonstrate here that sulfated oligosaccharide probes from heparin can be effectively and covalently attached without prior derivatization onto the hydrazide-modified, self-assembled monolayer on gold-coated slide surfaces in a microarray format. This platform is used to assess binding of specific heparin-binding protein targets at very high sensitivity, and we also demonstrate that the approach can be extended to nonsulfated sugars. Direct attachment of nonderivatized sugar probes on the chip is advantageous since it avoids the need for laborious prederivatization and cleanup steps. This versatile fluorescence microarray platform provides a facile approach for interrogating multiple carbohydrate-protein interactions in a high-throughput manner and has potential as a common gold surface platform for other diverse interrogations by MALDI-MS, surface plasmon resonance, and quartz crystal microbalances.

Multilayer thin films formed by sequential deposition of oppositely charged polypeptides on a charged surface are known from previous studies to comprise a mixture of types of secondary structure. Here, study of the perturbation of polypeptide film structure by deposition of poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonate) (PSS) on the film surface has revealed differences in behavior attributable to physical properties of the peptides. The methods of analysis were circular dichroism spectroscopy (CD), ultraviolet spectroscopy (UVS), and quartz crystal microbalance (QCM). Films made of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PLGA) with an average charge per monomer of about 1 were substantially more susceptible to perturbation of structure than films made of designed polypeptides with an average charge per monomer of about 0.5, despite preparation under identical conditions. PLL-PLGA films showed loss or gain of material and change in secondary structure content on perturbation, whether made of high molecular mass (ca. 90 kDa) or low molecular mass (ca. 14 kDa) polymers. By contrast, films made of very low molecular mass (ca. 3.5 kDa) designed polypeptides showed little change in secondary structure content. The data suggest that the penetrability of PSS or PAH into a film and therefore film density can depend substantially on the polypeptides of which it is made and the character of intermolecular interactions.

Micromachining techniques, which originated in the microelectronics industry, have been employed to manufacture microparticles bearing an engraved dot-type signature for biomolecular encoding. These metallic microstructures are photolithographically defined and manufactured in a highly reproducible manner. In addition, the code introduced on the particle face is a straightforward visible feature that is easily recognizable with the use of optical microscopy. The number of distinct codes theoretically could be many thousands, depending on the coding element numbers. Such microparticles are, thus, with appropriate surface organic functionalizations, ideal for encoding biomolecular libraries and serving as a platform for developing high-throughput multiplexed bioassay schemes based on suspension array technology. As proof of this statement, we demonstrated that encoded microparticles tagged with antibodies to human immunoglobulin classes are capable, using imaging detection as the interrogating approach, of high sensitivity and high specificity, as well as multiplexed detection of the respective antigens in a microliter-sample volume.