In situ generation of plasmonic cavities for high sensitivity fluorophore and biomolecule detection.

Plasmonic cavities are widely studied for the large amplifications in fluorophore emission intensity that they can achieve. Exploiting these properties for biological sensing applications requires strategies to selectively insert the target antigen into a resonant cavity, which are often of similar size or smaller than the target molecule. Here we demonstrate that using relatively simple solution processing, cavity structures can be grown at the stochastic locations where antigen binding takes place, which yields large enhancements in fluorophore emission intensities and over an order of magnitude improvement in bioassay response. The fluorescence amplification generated by the in situ growth of plasmonic structures is sufficiently large to enable both single antigen and single low-quantum efficiency fluorophore detection. The simplicity of the process demonstrated negates the requirement for complex surfaces or geometries and is readily adaptable for a wide range of diagnostic applications.

Controlled surface plasmon enhanced fluorescence from 1D gold gratings via azimuth rotations.

We demonstrate a method to maximise the fluorescence enhancement from a dye using gold coated diffraction gratings. Rotations about the azimuth provides a convenient approach to maximise the coupling between the grating and excitation source while achieving enhancements comparable to traditional optical configurations where the grating and in plane light vectors are parallel. This approach yields a 30 fold enhancement in the fluorescence signal over metal free substrates, while opening up the range of possible orientations and configurations suitable for fluorescence enhancement applications.

A catalyst-free and facile route to periodically ordered and c-axis aligned ZnO nanorod arrays on diverse substrates.

In this work we present a method for the deposition of periodically ordered, c-axis aligned ZnO nanorod arrays. By using chemical bath deposited films in conjunction with silica templating through nanosphere monolayers, masks suitable for high temperature deposition are created. A vapour phase transport technique is then used to deposit ordered arrays, quickly and inexpensively in a manner ideal for low cost, scalable and reproducible growth on a diverse range of substrates.

In vivo phage display to identify M cell-targeting ligands.

PURPOSE: The purpose of this study was to use in vivo phage display screening technology to identify novel lead peptides that target delivery to M cells and to follicle-associated epithelium (FAE) of the intestine. METHODS: Phage display libraries were screened in vivo within the gastrointestinal tract of a rat model by successive screenings across four cycles of selection. RESULTS: Following four cycles of in vivo screening, we identified 30 unique peptide sequences that bound to Peyer's patch tissue, human Caco-2, and rat IEC-6 epithelial cells. Two of the lead targeting peptides, peptides P8 (LETTCASLCYPS) and P25 (VPPHPMTYSCQY), were shown to bind to receptors on the surface of human intestinal tissue. The L-form, D-form, retro-inverted D-form, and selective Cys-to-Ala site-directed mutants of peptides P8 and P25 were also shown to retain binding to Caco-2 cell membranes when immobilized on the surface of a model particulate. Finally, the D-peptide analog of peptide P8 (yqcsytmphppv) enhanced the delivery of polystyrene particles to M cells in vivo in a mouse model, and these particles were delivered into Peyer's patch tissue, as determined by confocal microscopy. CONCLUSIONS: In summary, we have identified novel ligands that target M cells and Peyer's patch tissue, and thus may have utility in the targeted oral delivery of vaccines and vaccine carrier systems to the mucosal immune system within the gastrointestinal tract.

Cell culture modeling of specialized tissue: identification of genes expressed specifically by follicle-associated epithelium of Peyer's patch by expression profiling of Caco-2/Raji co-cultures.

Peyer's patch follicle-associated epithelium (FAE) regulates intestinal antigen access to the immune system in part through the action of microfold (M) cells which mediate transcytosis of antigens and microorganisms. Studies on M cells have been limited by the difficulties in isolating purified cells, so we applied TOGA mRNA expression profiling to identify genes associated with the in vitro induction of M cell-like features in Caco-2 cells and tested them against normal Peyer's patch tissue for their expression in FAE. Among the genes identified by this method, laminin beta3, a matrix metalloproteinase and a tetraspan family member, showed enriched expression in FAE of mouse Peyer's patches. Moreover, the C. perfringens enterotoxin receptor (CPE-R) appeared to be expressed more strongly by UEA-1(+) M cells relative to neighboring FAE. Expression of the tetraspan TM4SF3 gene and CPE-R was also confirmed in human Peyer's patch FAE. Our results suggest that while the Caco-2 differentiation model is associated with some functional features of M cells, the genes induced may instead reflect the acquisition of a more general FAE phenotype, sharing only select features with the M cell subset.

Peptidoglycan recognition protein expression in mouse Peyer's Patch follicle associated epithelium suggests functional specialization.

Mammalian Peyer's Patches possess specialized epithelium, the follicle associated epithelium (FAE), and specialized cells called M cells which mediate transcytosis of antigens to underlying lymphoid tissue. To identify FAE specific genes, we used TOGA gene expression profiling of microdissected mouse Peyer's Patch tissue. We found expression of laminin beta3 across the FAE, and scattered expression of peptidoglycan recognition protein (PGRP)-S. Using the M cell specific lectin Ulex europaeus agglutinin 1 (UEA-1), PGRP-S expression was nearly exclusively co-localized with UEA-1+ M cells. By contrast, the related gene PGRP-L was expressed among a subset of UEA-1 negative FAE cells. Expression of these proteins in transfected cells demonstrated distinct subcellular localization. PGRP-S showed a vesicular pattern and extracellular secretion, while PGRP-L showed localization to both the cytoplasm and the cell surface. The potential function of these PGRP proteins as pattern recognition receptors and their distinctive cellular distribution suggests a complex coordination among specialized cells of the FAE in triggering mucosal immunity and innate immune responses.

Catching target receptors for drug and vaccine delivery using TOGA gene expression profiling.

Drug delivery technologies are commonly directed towards formulations to control the delivery of therapeutic compounds. However, many processes in the human body have evolved to regulate the transport of various molecules, cells, or particles across epithelial barriers. To take advantage of this biology, we used TOGA gene expression profiling to identify receptor or transporter molecules to target delivery vehicles for transport across an epithelial barrier. In the case of intestinal epithelium, we sought molecules associated with the transport of particles by Peyer's patch M cells. We have identified genes specific to Peyer's patch epithelium, some of which appear to be M cell specific. Discoveries made by this process will provide targets for development of new vaccines, but also provide new insights into the biology of transepithelial transport. The power of this gene profiling approach also suggests application to other systems, such as the response to metabolic changes or drug treatments.