Pre-gastrula expression of zebrafish extraembryonic genes.

BACKGROUND: Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL). RESULTS: We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent. CONCLUSION: We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.

Three-dimensional tissue culture based on magnetic cell levitation.

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

Nanoshell-based substrates for surface enhanced spectroscopic detection of biomolecules.

Nanoshells are optically tunable core-shell nanostructures with demonstrated uses in surface enhanced spectroscopies. Based on their ability to support surface plasmons, which give rise to strongly enhanced electromagnetic fields at their surface, nanoshells provide simple, scalable, high-quality substrates. In this article, we outline the development and use of nanoshell-based substrates for direct, spectroscopic detection of biomolecules. Recent advances in the use of these nanostructures lead to improved spectroscopic quality, selectivity, and reproducibility.

Magnetic-plasmonic core-shell nanoparticles.

Nanoparticles composed of magnetic cores with continuous Au shell layers simultaneously possess both magnetic and plasmonic properties. Faceted and tetracubic nanocrystals consisting of wustite with magnetite-rich corners and edges retain magnetic properties when coated with a Au shell layer, with the composite nanostructures showing ferrimagnetic behavior. The plasmonic properties are profoundly influenced by the high dielectric constant of the mixed iron oxide nanocrystalline core. A comprehensive theoretical analysis that examines the geometric plasmon tunability over a range of core permittivities enables us to identify the dielectric properties of the mixed oxide magnetic core directly from the plasmonic behavior of the core-shell nanoparticle.

Real-time monitoring of lipid transfer between vesicles and hybrid bilayers on Au nanoshells using surface enhanced Raman scattering (SERS).

To investigate the dynamics of exchange/transfer of lipids between membranes, we have studied the interaction of donor-deuterated DMPC vesicles with DMPC hybrid bilayers on Au nanoshells using SERS. Experimental data confirm partial lipid exchange/transfer in the outer leaflet of the hybrid bilayer. The kinetics of the exchange/transfer process follows a first order process with a rate constant of 1.3 x 10(-4) s(-1). Changes in lipid phase behavior caused by the exchange/transfer process were characterized using generalized polarization measurements. In situ lipid transfer can potentially be utilized for preparation of asymmetric supported lipid bilayers and for incorporation of reporter lipids in biological membranes.

Interactions of ibuprofen with hybrid lipid bilayers probed by complementary surface-enhanced vibrational spectroscopies.

The incorporation of small molecules into lipid bilayers is a process of biological importance and clinical relevance that can change the material properties of cell membranes and cause deleterious side effects for certain drugs. Here we report the direct observation, using surface-enhanced Raman and IR spectroscopies (SERS, SEIRA), of the insertion of ibuprofen molecules into hybrid lipid bilayers. The alkanethiol-phospholipid hybrid bilayers were formed onto gold nanoshells by self-assembly, where the underlying nanoshell substrates provided the necessary enhancements for SERS and SEIRA. The spectroscopic data reveal specific interactions between ibuprofen and phospholipid moieties and indicate that the overall hydrophobicity of ibuprofen plays an important role in its intercalation in these membrane mimics.

Tailoring plasmonic substrates for surface enhanced spectroscopies.

Our understanding of how the geometry of metallic nanostructures controls the properties of their surface plasmons, based on plasmon hybridization, is useful for developing high-performance substrates for surface enhanced spectroscopies. In this tutorial review, we outline the design of metallic nanostructures tailored specifically for providing electromagnetic enhancements for surface enhanced Raman scattering (SERS). The concepts developed for nanoshell-based substrates can be generalized to other nanoparticle geometries and scaled to other spectroscopies, such as surface enhanced infrared absorption spectroscopy (SEIRA).

Nanoshells made easy: improving Au layer growth on nanoparticle surfaces.

The growth of a continuous, uniform Au layer on a dielectric nanoparticle is the critical step in the synthesis of nanoparticles such as nanoshells or nanorice, giving rise to their unique geometry-dependent plasmon resonant properties. Here, we report a novel, streamlined method for Au layer metallization on prepared nanoparticle surfaces using carbon monoxide as the reducing agent. This approach consistently yields plasmonic nanoparticles with highly regular shell layers and is immune to variations in precursor or reagent preparation. Single particle spectroscopy combined with scanning electron microscopy reveal that thinner, more uniform shell layers with correspondingly red-shifted optical resonances are achievable with this approach.

A cellular Trojan Horse for delivery of therapeutic nanoparticles into tumors.

Destruction of hypoxic regions within tumors, virtually inaccessible to cancer therapies, may well prevent malignant progression. The tumor's recruitment of monocytes into these regions may be exploited for nanoparticle-based delivery. Monocytes containing therapeutic nanoparticles could serve as "Trojan Horses" for nanoparticle transport into these tumor regions. Here we report the demonstration of several key steps toward this therapeutic strategy: phagocytosis of Au nanoshells, and photoinduced cell death of monocytes/macrophages as isolates and within tumor spheroids.

Electromigrated nanoscale gaps for surface-enhanced Raman spectroscopy.

Single-molecule detection with chemical specificity is a powerful and much desired tool for biology, chemistry, physics, and sensing technologies. Surface-enhanced spectroscopies enable single-molecule studies, yet reliable substrates of adequate sensitivity are in short supply. We present a simple, scaleable substrate for surface-enhanced Raman spectroscopy (SERS) incorporating nanometer-scale electromigrated gaps between extended electrodes. Molecules in the nanogap active regions exhibit hallmarks of very high Raman sensitivity, including blinking and spectral diffusion. Electrodynamic simulations show plasmonic focusing, giving electromagnetic enhancements approaching those needed for single-molecule SERS.

Chain-length-dependent vibrational resonances in alkanethiol self-assembled monolayers observed on plasmonic nanoparticle substrates.

Alkanethiol self-assembled monolayers (SAMs) on gold exhibit a series of sharp resonances in their surface-enhanced Raman spectrum that depend dramatically on carbon chain length. This unusual behavior suggests a coupling of the gold-sulfur bond stretch with the longitudinal acoustic, "accordion", vibrations of the molecular alkane chain. A simple model of a one-dimensional chain attached to a surface quantitatively reproduces these previously unreported experimental observations in this important nanomaterial system.

In vivo detection of gold-imidazole self-assembly complexes: NIR-SERS signal reporters.

Here we report in vitro and in vivo detection of self-assembled Au-imidazole by using near-infrared surface-enhanced Raman scattering (NIR-SERS). In vivo, the Au-imidazole structures were administered into tumor-bearing mice and detected noninvasively. The self-assembled Au-imidazole complexes were generated by the adsorption of imidazole molecules onto Au nanoparticles (NP) and were then characterized as aqueous suspensions by using NIR-SERS, angle-dependent light scattering with fractal dimension analysis, and visible extinction spectroscopy. The structure and optical activity was sensitive to imidazole concentration and Au NP size. Specifically, the Au-imidazole assemblies formed at lower imidazole concentrations had the lowest fractal dimension (D(f) = 1.2) and the largest Raman enhancement factors for the dominant NIR-SERS feature, a ring-breathing vibrational mode at 954 cm(-1). Changes in elastic scattering intensity, fractal dimension, and surface plasmon absorption were observed with increasing imidazole concentration. The Raman enhancement factor was also found to range between 10(6) and 10(9) with different primary Au nanoparticle sizes. For the higher enhancement factor systems, NIR-SERS detection of Au-imidazole was performed with data acquisitions time of only 5 s. The largest enhancement was observed for the 954-cm(-1) feature at an imidazole concentration of 1.9 microM when coupled to 54-nm-diameter Au NPs (the largest NP tested). Finally, we show the first demonstration of in vivo, noninvasive, and real-time SERS detection.

All-optical nanoscale pH meter.

We show that an Au nanoshell with a pH-sensitive molecular adsorbate functions as a standalone, all-optical nanoscale pH meter that monitors its local environment through the pH-dependent surface-enhanced Raman scattering (SERS) spectra of the adsorbate molecules. Moreover, we also show how the performance of such a functional nanodevice can be assessed quantitatively. The complex spectral output is reduced to a simple device characteristic by application of a locally linear manifold approximation algorithm. The average accuracy of the nano-"meter" was found to be +/-0.10 pH units across its operating range.

Determining the conformation of thiolated poly(ethylene glycol) on Au nanoshells by surface-enhanced Raman scattering spectroscopic assay.

The packing density of thiolated poly(ethylene glycol) (PEG) adsorbates on Au nanoshells is determined by exploiting the surface-enhanced Raman scattering response of individual nanoshell substrates. By incorporating the linker molecule p-mercaptoaniline (pMA), the number of 2000 MW and 5000 MW PEG molecules on each nanoparticle is determined by interpolation of the Langmuir isotherm for pMA. We conclude that both PEG adsorbates maintain a compact "brush" rather than an extended "mushroom" configuration on nanoshell surfaces.

Nanosphere arrays with controlled sub-10-nm gaps as surface-enhanced raman spectroscopy substrates.

We demonstrate a convenient and cost-effective chemical approach for fabricating highly ordered Au nanoparticle arrays with sub-10-nm interparticle gaps. Near-field enhancements inside the interparticle gaps create uniform periodic arrays of well-defined "hot spots" exploitable for large surface-enhanced Raman spectroscopy (SERS) enhancements. A cetyltrimethylammonium bromide (CTAB) bilayer surrounding each individual nanoparticle upon array crystallization is responsible for this periodic gap structure; displacement of the CTAB by smaller thiolated molecules does not affect the structural integrity of the arrays. As SERS substrates, the as-fabricated Au nanoparticle arrays exhibit high SERS sensitivity, long-term stability, and consistent reproducibility.