Assembly of bioactive multilayered nanocoatings on pancreatic islet cells: incorporation of α1-antitrypsin into the coatings.

A spontaneous multilayer deposition approach for presenting therapeutic proteins onto pancreatic islet surfaces, using a heparin polyaldehyde and glycol chitosan alternating layering scheme, has been developed to enable the nanoscale engineering of a microenvironment for transplanted cells. The nanocoating incorporating α1-antitrypsin, an anti-inflammatory protein, exhibited effective anti-coagulant activities in vitro.

Nanolayer encapsulation of insulin-chitosan complexes improves efficiency of oral insulin delivery.

Current oral insulin formulations reported in the literature are often associated with an unpredictable burst release of insulin in the intestine, which may increase the risk for problematic hypoglycemia. The aim of the study was to develop a solution based on a nanolayer encapsulation of insulin-chitosan complexes to afford sustained release after oral administration. Chitosan/heparin multilayer coatings were deposited onto insulin-chitosan microparticulate cores in the presence of poly(ethylene) glycol (PEG) in the precipitating and coating solutions.

Multilayered nanocoatings incorporating superparamagnetic nanoparticles for tracking of pancreatic islet transplants with magnetic resonance imaging.

A novel strategy for delivering functionalised superparamagnetic iron oxide nanoparticles to the outer surface of pancreatic islet grafts, using chemically modified polymeric nanolayers, has been developed for tracking of engrafted pancreatic islets by magnetic resonance imaging.

Fluorescence intensity- and lifetime-based glucose sensing using glucose/galactose-binding protein.

We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing.

Multilayer nanoencapsulation: a nanomedicine technology for diabetes research and management.

Nanothickness encapsulation using a layer-by-layer technique has applications in several areas of diabetes research, including improved glucose sensors, islet cell transplantation and oral insulin delivery. We have fabricated microvesicles containing a fluorescence lifetime-based glucose sensing system, with bacterial glucose-binding protein as the glucose receptor. Such sensors are suitable for impregnation in the dermis as a 'smart tattoo' type of non-invasive glucose monitoring technology.

Improving cellular function and immune protection via layer-by-layer nanocoating of pancreatic islet β-cell spheroids cocultured with mesenchymal stem cells.

Islet transplantation as a therapy for type 1 diabetes is currently limited by lack of primary transplant material from human donors and post-transplantation loss of islets caused by adverse immune and nonimmune reactions. This study aimed to develop a novel strategy to create microenvironment for islets via integration of nanoencapsulation with cell cocultures, thereby enhancing their survival and function. The nanoencapsulation was achieved via layer-by-layer deposition of phosphorycholine-modified poly-L-lysine/heparin leading to the formation of nanometer-thick multilayer coating on islets.

Polysaccharide multilayer nanoencapsulation of insulin-producing beta-cells grown as pseudoislets for potential cellular delivery of insulin.

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.

Fluorescence lifetime spectroscopy and imaging of nano-engineered glucose sensor microcapsules based on glucose/galactose-binding protein.

We aimed to develop microsensors for eventual glucose monitoring in diabetes, based on fluorescence lifetime changes in glucose/galactose-binding protein (GBP) labelled with the environmentally sensitive fluorophore dye, badan. A mutant of GBP was labelled with badan near the binding site, the protein adsorbed to microparticles of CaCO(3) as templates and encapsulated in alternating nano-layers of poly-L-lysine and heparin. We used fluorescence lifetime imaging (FLIM) with two-photon excitation and time-correlated single-photon counting to visualize the lifetime changes in the capsules.

Saccharide microarrays for high-throughput interrogation of glycan-protein binding interactions.

This chapter describes two methods for fabricating microarrays of saccharides for display and interrogation with binding proteins, using fluorescence detection. The first approach is based on the rapid immobilization of heparan sulphate glycans upon commercially available aminosilane slides via their reducing ends. The second approach is based on the use of a hydrazide-derivatized self-assembled monolayer (SAM) on a gold-coated slide surface.

Nanomedicine and its potential in diabetes research and practice.

Nanomedicine involves measurement and therapy at the level of 1-100 nm. Although the science is still in its infancy, it has major potential applications in diabetes. These include solving needs such as non-invasive glucose monitoring using implanted nanosensors, with key techniques being fluorescence resonance energy transfer (FRET) and fluorescence lifetime sensing, as well as new nano-encapsulation technologies for sensors such as layer-by-layer (LBL) films.

Towards GAG glycomics: analysis of highly sulfated heparins by MALDI-TOF mass spectrometry.

Glycomics is a developing field that provides structural information on complex populations of glycans isolated from tissues, cells and organs. Strategies employing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are central to glycomic analysis. Current MALDI-based glycomic strategies are capable of efficiently analyzing glycoprotein and glycosphingolipid glycomes but little attention has been paid to devising glycomic methodologies suited to the analysis of glycosaminoglycan (GAG) polysaccharides which pose special problems for MALDI analysis because of their high level of sulfation and large size.

Fabrication of carbohydrate microarrays on gold surfaces: direct attachment of nonderivatized oligosaccharides to hydrazide-modified self-assembled monolayers.

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.

Straightforward and effective protein encapsulation in polypeptide-based artificial cells.

A simple and straightforward approach to encapsulating an enzyme and preserving its function in polypeptide-based artificial cells is demonstrated. A model enzyme, glucose oxidase (GOx), was encapsulated by repeated stepwise adsorption of poly(L-lysine) and poly(L-glutamic acid) onto GOx-coated CaCO3 templates. These polypeptides are known from previous research to exhibit nanometer-scale organization in multilayer films.

High-capacity functional protein encapsulation in nanoengineered polypeptide microcapsules.

Addition of polyethylene glycol to aqueous assembly solutions of oppositely charged polypeptides enables high-capacity "loading" of functional protein in biocompatible microcapsules by template-supported layer-by-layer nanoassembly.

Perturbation of nanoscale structure of polypeptide multilayer thin films.

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).

Microfabrication of encoded microparticle array for multiplexed DNA hybridization detection.

A strategy for the high-sensitivity, high-selectivity, and multiplexed detection of oligonucleotide hybridizations has been developed with an encoded Ni microparticle random array that was manufactured by a "top-down" approach using micromachining and microfabrication techniques.

Electrocatalytic activity of DNA on electrodes as an indication of hybridisation.

Electron transfer between metal electrodes and ferro/ferricyanide is completely suppressed at low ionic concentration. We describe here a new phenomenon related to this reaction: an immobilisation of thiolated single-stranded DNA on gold electrodes retains this activity at low ionic strength up to the level corresponding to the high ionic strength. In contrast, a hybridisation of the complementary DNA with the thiolated single-stranded DNA followed by a binding onto the electrodes, attenuated the electrocatalytic effect.

Nanosystems for biosensing: multianalyte immunoassay on a protein chip.

This chapter describes the construction of addressable two-dimensional (2D) microarrays via the random fluidic self-assembly of metallic particles and the use of these arrays as platforms for constructing protein chips for bioassays. These arrays will be useful as platforms for constructing protein chips for bioassays in a broad range of applications. The basic units in the assembly are microfabricated particles, which carry a straightforward visible code, and the corresponding array template patterned on a glass substrate.

Micromachining microcarrier-based biomolecular encoding for miniaturized and multiplexed immunoassay.

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.

Screening of clostebol and its metabolites in bovine urine with ELISA and comparison with GC-MS results in an interlaboratory study.

An extraction procedure for clostebol metabolites in urine is developed including enzymatic hydrolysis of conjugated metabolites with Helix pomatia juice (SHP) and solid-phase extraction (SPE) with further cleanup of sample extracts. For the enzymatic deconjugation step, variables such as buffer pH, amount of enzyme, incubation time, and temperature are examined. For the SPE step, different wash solutions and combinations with subsequent liquid-liquid extractions are examined.

Multianalyte immunoassay with self-assembled addressable microparticle array on a chip.

This paper describes the random fluidic self-assembly of metallic particles into addressable two-dimensional microarrays and the use of these arrays as a platform for constructing a biochip useful for bioassays. The basic units in the assembly were the microfabricated particles carrying a straightforward visible code and the corresponding array template patterned on a glass substrate. The particles consisted of a hydrophobic and magnetic Ni-polytetrafluoroethylene (PTFE) composite layer on one face, and on the other face a gold layer that was modified for biomolecular attachment.