N A N O P R O B E S     E - N E W S

Vol. 7, No. 6          June 15, 2006

Nanogold labeling was complemented by the novel ReAsH detection system, in which a helical tetracysteine expression tag is bound highly selectively with a biarsenical compound which becomes fluorescent upon binding. This label also provides a method for brightfield light and electron microscopy: it may be photooxidized, then treated with diaminobenzidine (DAB) followed by osmium tetroxide to give a continuous dark stain. The authors use ReAsH-DAB-osmium tetroxide to determine the distribution of tetracysteine-tagged HLA-G, expressed in HLA-Gtransduced INS cells. While ReAsH-DAB-osmium staining, because of its more continuous nature, provides a highly effective means for identifying organelles in which the target is localized; labeling with Nanogold and counting of the gold-enhanced particles within specific organelle volumes afforded quantitative comparison between the labeling density within organelles, and the size and distribution of the target organelles with the cells, allowing a distinction to be made between extent and labeling density.

The clear visualization afforded by gold enhancement, a novel autometallographic procedure in which immunogold labels are enlarged by the deposition of gold rather than silver, was used to quantitate the labeling in different cellular compartments. Gold enhancement has significant advantages for both scanning electron microscopy (SEM) and transmission electron microscopy (TEM):

• Gold enhancement may safely be used before osmium tetroxide - it is not etched.
• May be used in physiological buffers (chlorides precipitate silver, but not gold).
• The metallographic reaction is less pH sensitive than that of silver.
• Gold gives a much stronger backscatter signal than silver.
• GoldEnhance is near neutral pH for best ultrastructural preservation.
• Low viscosity, so the components may be dispensed and mixed easily and accurately.

HLA-G was localized at the ultrastructural level using in the INS endocrine beta-cell line. First, tetracysteine-tagged HLA-G, expressed in HLA-Gtransduced INS cells, was labeled with ReAsH-EDT₂. The cells were then fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 and the ReAsH label was visualized by photoconversion. 1 mg/ml diaminobenzidine in oxygenated 0.1 M sodium cacodylate buffer was added to the dish, and the cells exposed to a 585-nm light from a xenon lamp until a brownish reaction product appeared. After postfixing in 1% osmium tetroxide, the cells were dehydrated and embedded in Epon 812. Ultrathin sections were examined at a Jeol-1200 electron microscope. This approach confirmed that a predominant intracellular location of the protein is in the endoplasmic reticulum, the main cellular compartment where class I major histocompatibility complex (MHC) molecules are synthesized and assembled; a patchy expression of HLA-G was also detected at the basolateral but not at the apical side of the cell membrane, indicating targeting of this molecule to distinct membrane microdomains of beta-cells. Photo-oxidation also revealed membrane labeling of some insulin-containing granules.

The distribution of native protein was then quantitated using Nanogold with gold enhancement. HLA-Gtransduced INS or MIN-6 cells, grown on glass coverslips, were fixed in 4% paraformaldehyde, 0.05% glutaraldehyde, 1% l-lysine, and 0.25% sodium metaperiodate in 0.04 M sodium cacodylate buffer at pH 7.4, then permeabilized with 0.1% saponin. After washing and blocking, samples were incubated overnight at 4°C with primary antibody (mAb 4H84, 2.5 µg/ml) followed by Nanogold goat anti-mouse Fab fragments. After washing, samples were fixed in 1.2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, with 5% sucrose. The Nanogold particles were enhanced using GoldEnhance LM. After postfixation in 2% osmium tetroxide and 2% potassium ferricyanide, they were dehydrated, embedded in Epon 812, sectioned, and stained with lead citrate.

For morphometric analysis, the volume density of secretory granules was evaluated by planimetry, and the number of gold particles over secretory granules and over the whole cell were scored. The relationship between the number of gold particles in granules and the volume density of these organelles was analyzed by regression analysis, and the theoretical distribution of gold particles over granules, as predicted by random labeling, was compared with the distribution actually observed using a chi-squared test. The results showed there was no correlation between levels of HLA-G expression in the cell and frequency of HLA-G⁺ granules. Within individual cells, the proportion of gold particles over secretory granules was larger than the relative volume occupied by these organelles in the cells. No significant correlation was observed between the volume density of secretory granules and the amount of gold labeling of these organelles (r² = 0.005, P = 0.58), indicating that the gold labeling did not merely reflect the relative abundance of granules in the cells. In contrast, a highly significant difference was observed between the distribution of granule immunolabeling and that computed assuming a random staining (P = 0.001), indicating that the gold labeling in granules was specific.

Surface expression of this HLA determinant in endocrine cells is regulated in response to growth and inflammatory stimuli. Comparison of the observed distribution with immunofluorescent studies and other data suggests that HLA-G expressed in this tissue may associate with a subset of insulin-containing granules and may be shuttled to the cell surface in response to secretory stimuli. Many autoantigens in islet immunity are components of secretory granules; it is possible that sites of insulin exocytosis may represent subcellular domains where a high density of potentially immunogenic ligands become exposed. Local clustering of immunoregulatory molecules, such as HLA-G, at sites of granule exocytosis may prevent the unwanted activation of autoreactive T-cells, and HLA-G presentation by endocrine cells may be regulated in concert with their secretory activity. These results identify the expression of a major histocompatibility complex locus with putative regulatory functions in human pancreatic islets; this finding has potentially important implications for the progression of autoimmunity, as well as for the establishment of transplant tolerance to this tissue.

Reference:

Cirulli, V.; Zalatan, J.; McMaster, M.; Prinsen, R.; Salomon, DR.; Ricordi, C.; Torbett, BE.; Meda, P., and Crisa, L.: The class I HLA repertoire of pancreatic islets comprises the nonclassical class Ib antigen HLA-G. Diabetes, 55, 1214-1222 (2006).

• Abstract (courtesy of Diabetes).

More information:

• Nanogold Conjugates: catalog and general information
• Nanogold Conjugates: technical help
• Nanogold-antibody Conjugates: product information and instructions
• Gold Enhancement: general and catalog information
• Gold Enhancement: technical help
• Gold Enhancement: technical help
• GoldEnhance: obtaining the cleanest signal

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Labeling Aromatic Groups

Aromatic Amines

Aromatic amines are much weaker bases than their aliphatic counterparts. They are not usually protonated at the pH ranges used for labeling aliphatic amines with Mono-Sulfo-NHS-Nanogold®, and are much less reactive. More forcing conditions, such as higher pH values (pH 9.0 - 10) are usually not sufficient for reaction. Chemically, this is because reaction proceeds via nucleophilic attack of the lone pair of electrons on the amino- nitrogen atom on the Sulfo-NHS-ester, displacing the NHS group to form an amide bond. However, in aromatic amines, this lone pair is partially conjugated with the aromatic pi-system, and much less available for reaction. Therefore, reaction with NHS esters is more difficult. As a result, while aliphatic amines will react if the pH is simply kept high enough to prevent protonation of the amine to the ammonium ion R-NH₃⁺, in order to react directly, aromatic amines must be partially deprotonated so that there is a contribution from the Ar-NH^- form in order to facilitate reaction. This requires more forcing conditions, and usually a much stronger base.

To conduct such labeling reactions, we suggest the following strategy:

• Conduct the reaction in an aprotic organic solvent rather than in water, aqueous buffers or alcohols. Dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMA) make good choices because they are good solvents for Nanogold and are often compatible with water-soluble molecules.

• You will need a strong, hindered base. Triethylamine is a good choice to try first: add a couple of drops of triethylamine to 1 mL of DMSO or DMA solution of the compound you wish to label, then add the Mono-Sulfo-Nanogold, and stir at room temperature for one hour. Use an excess of a smaller molecule, but use an excess of Nanogold if you are labeling a molecule that is larger than Nanogold.

• If the reaction is complete, you can transfer the reaction mixture to aqueous or alcoholic solution, then separate the conjugate from unconjugated Nanogold or any excess of the molecule you are labeling using gel filtration or other usual methods.

• Test the conjugate for reactivity. We recommend either:
  • A blot test, in which the target is spotted in serial dilutions onto a nitrocellulose membrane, then detected by application of the Nanogold conjugate followed by silver enhancement or gold enhancement blot, or;

  • If the probe target is larger than the probe, you can mix the target with the labeled probe in solution, then separate the mixture using a size fractionation column such as Pharmacia Superose-6 or Superose-12. If binding occurs, a large labeled species will elute, and you will observe little or no peak for free Nanogold; if it does not, then the target and labeled probe will elute separately.

• If this does not produce labeling, consider a stronger hindered base, such as LHMDS (lithium hexamethyldisilylamine) or lithium diisopropylamide. However, these should be used cautiously, since they are much stronger than triethylamine and may produce undesirable reactions elsewhere in your molecule.

You should consider an alternative approach: use a more reactive compound to convert the aromatic amine to a species that will react more easily with one of our Nanogold labeling reagents. More reactive species such as isocyanates, isothiocyanates, sulfonyl chlorides, or carboxylic acid chlorides, may be used to introduce a carboxylic acid or carboxylic ester; once hydrolyzed to the free acid, this may be converted to an activated aliphatic ester - for example, using EDC and Sulfo-NHS, as in peptide synthesis - and reacted with Monoamino Nanogold. Alternatively, the amine can be reacted with 2-iminothiolane (Traut's reagent) to introduce an aliphatic thiol that may then be labeled with Monomaleimido Nanogold. These reactions are shown below.

Aromatic Carboxylates

carboxylic acids and esters are actually a preferred route to labeling aromatic entities. This is because their reactivity is very similar to that of their aliphatic counterparts, and is even enhanced by the electron-withdrawing effect of the aromatic group. We have previously discussed strategies for labeling carboxylic acids, and these reactions, using Monoamino Nanogold® with either EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride)/Sulfo-NHS, or (b) 1,1-carbonyl-diimidazole (CDI), shown below, are well suited to labeling aromatic carboxylic acids:

• The reaction used in peptide synthesis usually works well: activate the carboxylic acid with EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide Hydrochloride) and Sulfo-NHS to convert it to a reactive Sulfo-N-hydroxysuccinimide ester, then react with Monoamino Nanogold. You can purchase EDC from a number of sources; its use is described by Pierce. EDC reacts with the carboxyl groupto form an amine-reactive O-acylisourea intermediate. This intermediate could then react with Monoamino Nanogold; however, it is also susceptible to hydrolysis, making it unstable and short-lived in aqueous solution. The addition of Sulfo-N-hydroxysuccinimide (5 mM) stabilizes the amine-reactive intermediate by converting it to an amine-reactive Sulfo-NHS ester, thus increasing the efficiency of EDC-mediated coupling reactions. The amine-reactive Sulfo-NHS ester intermediate has sufficient stability to permit two-step cross-linking procedures, which allow the carboxyl groups on one protein to remain unaltered.

  Reference:

  Staros, J. V.; Wright, R. W., and Swingle, D. M.: Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem., 156, 220-222 (1986).

  • Abstract (courtesy of PubMed (Medline)).

• Another reagent, that works well in non-aqueous systems, is 1,1-carbonyl-diimidazole (CDI). The molecule to be labeled should be dissolved in a small amount of the organic solvent and a small (5-fold to 10-fold) excess of CDA added; the pH is then raised to 7.5 or higher by the addition of aqueous reaction buffer, and the Monoamino Nanogold added. You should consider this reaction if you are labeling a molecule that is sensitive to water.

  Reference:

  Staab, H. A., and Rohr; W., in Newer Methods Prep. Org. Chem., 5, 61-108 (1968).

Aromatic Thiols

Aromatic thiols are rarely encountered in biological systems. However, their reactivity is similar to that of their aliphatic counterparts, and most will still react with Monomaleimido Nanogold. However, as with aromatic amines, the nucleophilicity of the sulfur atom is lower. More forcing conditions may therefore he required for reaction, such as gentle stirring for several hours at room temperature rather than incubation overnight at 4°C.

If you need help in finding a suitable synthesis, we would be glad to advise. Contact us with your questions.

More information:

• Guide to Gold Cluster Labeling
• Tech help: Nanogold labeling reagents
• Catalog information: Nanogold labeling reagents
• Product instructions: Monomaleimido Nanogold
• Product instructions: Mono-Sulfo-NHS-Nanogold
• Product instructions: Monoamino Nanogold
• References: Nanogold labeling reagents

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Pre-embedding Nanogold® Labeling Localizes Cannabinoid Receptors

For immunohistochemistry, anesthetized mice were fixed transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.2, for light microscopy, or 4% paraformaldehyde and 0.1% glutaraldehyde in PB for electron microscopy. 50 µm microslicer sections were prepared and incubated successively with 10% normal donkey serum, rabbit DAGLalpha antibody (0.52 µg/ml), biotinylated secondary antibody, and streptavidinperoxidase complex for 30 minutes using a Histofine SAB-PO(R) kit (Nichirei, Tokyo, Japan), followed by visualization using a tyramide signal amplification kit (TSA-DIRECT (Red)). Immunoreaction was visualized with 3,3'-diaminobenzidine.

For preembedding immunogold electron microscopy, microslicer sections were dipped in 5% BSA/0.02% saponin/PBS for 30 min, and incubated overnight with rabbit DAGLalpha (0.52 µg/ml), guinea pig CB1 (1 µg/ml), or rabbit PKCgamma (1 µg/ml) antibody diluted with 1% BSA/0.004% saponin/PBS and then with Nanogold IgG anti-rabbit IgG for two hours, followed by intensification with a HQ silver enhancement kit. Sections labeled by immunoperoxidase and silver-enhanced immunogold were treated with 2% osmium tetroxide for 30 minutes, stained in block with 2% uranyl acetate for 30 minutes, dehydrated, and embedded in Epon 812. For postembedding immunogold electron microscopy, microslicer sections were cryoprotected with 30% sucrose/PB and frozen rapidly with liquid propane. Frozen sections were immersed in 0.5% uranyl acetate in methanol at -90°C in a freeze-substitution unit, infiltrated at -45°C with Lowicryl HM-20 resin and polymerized with UV light. After etching with saturated sodium-ethanolate solution for 3 seconds, ultrathin sections on nickel grids were treated successively with 2% human serum albumin / 0.1% Tween 20 in Tris-buffered saline, pH 7.5 (HTBST) for 30 minutes, rabbit DAGLalpha antibody (15 µg/ml) in HTBST overnight, and 10 nm colloidal gold-labeled anti-rabbit IgG (1:100) in HTBST for 2 hours. Grids were stained with 2% uranyl acetate for 20 minutes and mixed lead solution for 30 seconds. For quantitative analysis, silver-enhanced immunogold particles for DAGLalpha on somatodendritic and spine membranes or for CB1 on parallel fibers were counted on electron micrographs. Tangential and perpendicular distributions of DAGLalpha in spines were examined from 57 spines of parallel fiberPurkinje cell synapses (postembedding immunogold) and 49 spines of CA1 pyramidal cell synapses (preembedding immunogold) and analyzed using IPLab software (Nippon Roper, Tokyo, Japan).

In the cerebellum, DAGLalpha was predominantly expressed in Purkinje cells. DAGLalpha was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGLalpha was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck, but was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGLalpha was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGLalpha was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGLalpha is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. The different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled, depending on neuron types.

Reference:

Yoshida T, Fukaya M, Uchigashima M, Miura E, Kamiya H, Kano M, Watanabe M.: Localization of diacylglycerol lipase-alpha around postsynaptic spine suggests close proximity between production site of an endocannabinoid, 2-arachidonoyl-glycerol, and presynaptic cannabinoid CB1 receptor. J. Neurosci., 26, 4740-4751 (2006).

• Abstract (courtesy of the Journal of Neuroscience).

More information:

• Preembedding Nanogold Labeling with HQ Silver enhancement: special feature
• Nanogold conjugates: catalog information
• Nanogold conjugates: technical help
• More Nanogold pre-embedding labeling references
• Silver enhancement: catalog information
• Silver enhancement: technical help
• Silver enhancement references, sorted by application

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Post-Embedding Nanogold® Labeling of Siliceous Spicules in Desmosponges

Sponges (phylum Porifera) of the class of Demospongiae are stabilized by a siliceous skeleton, composed of silica needles (spicules) which provide the morphogenetic scaffold of these metazoans. The center of the spicules contains an axial filament consisting predominantly of silicatein, an enzyme that catalyzes the synthesis of biosilica. The authors used differential display of transcripts to identify additional proteins involved in silica formation. Two genes were isolated from the marine desmosponge Suberites domuncula, coding for a galectin and for a fibrillar collagen. Galectin forms aggregates to which silicatein molecules bind, and association strongly increases the extent of the silicatein-mediated silica formation.

A new, mild extraction procedure was used to isolate axial filaments from spicules of S. domuncula, which was found to allow localization of these additional proteins. Previously, axial filaments had been obtained by HF treatment of isolated spicules; the milder procedure comprised treatment with lysis buffer supplemented with 4 M urea. Spicules were isolated from tissue, collected, and pulverized thoroughly in a mortar. The lysis-urea buffer was added, and the suspension stirred for 1 hour at 4°C then centrifuged at 10,000 X g for 5 minutes at 4°C. The supernatant was used for further analysis by SDS-PAGE and Western blotting.

Recombinant S. domuncula Galectin-2 was expressed in Escherichia coli from the complete open reading frame of SDGALEC2, using the glutathione S-transferase (GST) fusion system in the pET-41a(+) vector; the GST fusion protein (65 kDa) was purified by GST-Bind affinity chromatography and cleaved with enterokinase to separate the N-terminal GST tag and His tag as well as the S-tag from the recombinant galectin (rGALEC2_SUBDO). This resulting galectin-2 carried at its C terminus a small His tag. Polyclonal antibodies (pAb) were then raised against the purified, recombinant galectin-2 (rGALEC2_SUBDO) in female rabbits (New Zealand White); the pAb against galectin-2 was termed pAb-aGALEC2. The titer of the antibodies was 1:3,000. In the controls, adsorbed pAb-aGALEC2 (100 µl of antibodies were incubated with 20 µg of rGALEC2_SUBDO) was used. Polyclonal antibodies (pAbs) against silicatein (pAbaSILIC) were raised against axial filaments from spicules, containing mainly silicatein.

Electron immunogold labeling was performed with primmorph/tissue samples treated in glutaraldehyde/paraformaldehyde buffered in phosphate, pH 7.4. The material was dehydrated in ethanol and embedded in LR-White resin. 60-nm thick slices were cut and blocked with bovine serum albumin in PBS, then incubated with the primary antibody pAb-aSILIC (1:1,000) for 12 h at 4°C. In controls, preimmune serum was used. After three washes with PBS containing 1% BSA, sections were incubated with a 1:100 dilution of the Nanogold-IgG anti-rabbit, diluted 1:200, for 2 hours. Sections were rinsed in PBS, treated with glutaraldehyde/PBS, washed, and dried. Subsequently, the immunocomplexes were silver enhanced using the Danscher procedure.

In addition to silicatein, the axial filaments were found to contain galectin and a few other proteins. Blotting and immunoprecipitation studies on proteins extracted from primmorphs incubated for 05 days in seawater supplemented with 60 µM sodium silicate showed a strong increase in galectin expression after 2 - 5 days and indicated that galectin binds to silicatein. Initial synthesis of spicules occurs intracellularly in the sclerocytes; after reaching a critical length of 510 µm, the spicules are extruded into the bulky extracellular matrix within the mesohyl. Transmission electron microscopy showed that in this space, the spicules are surrounded by cells that never come closer than 10 nm; some of these 10-nm spaces contain string- and net-like structures. Immunogold electron microscopic studies demonstrated that pAb-aSILIC recognized aggregates of silicatein of different sizes, which were always associated with the string- and net-like structures. The presence of galectin in the axial canal and on the surface of the spicules, colocalized with silicatein, was demonstrated by immunofluorescence. When associated with collagen fibers, these silicatein/string- and net-like structures were found to be organized in concentric rings, suggesting sequential association of the three components, silicatein (as immunogold signal), string-/net-like structures, and collagen. In the initial phase the monomeric gold particles are arranged (in a "pearl string" manner) in double rows.

These results suggest that collagen fibers mediate the functional orientation of silicatein-galectin-2 complexes by arranging them concentrically around the longer axis of a growing spicule. Then, silicatein mediates silica deposition onto the surface of the existing silica layer. Because the surface of a new siliceous spicule is also covered with silicatein, the appositional growth/thickening of a spicule proceeds from two directions (centrifugal and centripetal). Galectin, in addition to silicatein, presumably forms in the axial canal as well as on the surface of the spicules an organized net-like matrix. Taken together, these additional proteins, working together with silicatein, may also be relevant for potential (nano)-biotechnological applications of silicatein in the biosynthesis of silica and the formation of surface coatings.

Reference:

Schröder, H. C.; Boreiko, A.; Korzhev, M.; Tahir, M. N.; Tremel, W.; Eckert, C.; Ushijima, H.; Müller, I. M., and Müller, W. E.: Co-expression and functional interaction of silicatein with galectin: matrix-guided formation of siliceous spicules in the marine demosponge Suberites domuncula. J. Biol. Chem., 281, 12001-12009 (2006).

• Abstract (courtesy of the Journal of Biological Chemistry).

More information:

• Nanogold conjugates: product information and instructions
• Nanogold conjugates: catalog information
• Nanogold post-embedding labeling: other references
• Nanogold references sorted by application

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nanoprobes at Microscopy and Microanalysis 2006

Our paper, number 815, entitled "Light and Electron Microscopy of Microsporida using Enzyme Metallography," will be given at 9:15 am as a platform presentation in session B05, "Advances in Correlative and High Resolution Labeling," scheduled for Wednesday, August 2, beginning 8:15 am in room 310/312. Microscopy & Microanalysis 2006 will be held in the convention facilities on Chicago's Navy Pier.

More information:

• Microscopy & Microanalysis 2006
• Microscopy & Microanalysis 2006 - Abstract search engine
• Enzyme metallography - paper from Microscopy & Microanalysis 2002
• Enzyme metallography - paper from Microscopy & Microanalysis 2004
• Enzyme metallography for in situ hybridization
• Enzyme metallography for immunohistochemistry
• Validation of a dual ISH (enzyme metallography) and IHC HER2 assay

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Other Recent Publications

Reference:

Stoeva, S. I.; Lee, J. S.; Thaxton, C. S., and Mirkin, C. A.: Multiplexed DNA Detection with Biobarcoded Nanoparticle Probes. Angew. Chem. Int. Ed. Engl., 45, 3303-3306 (2006).

• Abstract (courtesy of Wiley Interscience).

It was a good month for hybrid nanoparticles - nanoparticles containing two types of metal domains. Pellegrino and co-workers describe, in the Journal of the American Chemical Society, a procedure for preparing colloidal nanocrystal CoPt₃ - Au dimers. Dimers of CoPt₃-Au were prepared by nucleation of gold on preformed CoPt₃ nanoparticles. Typically, a sonicated solution of AuCl₃, dodecylethyl-dimethylammonium bromide (DEDAB, which forms a soluble complex with AuCl₃), and dodecylamine in a molar ratio of 1 : 1.8 : 8 in toluene was added dropwise to a colloidal solution of CoPt₃ nanoparticles kept under inert atmosphere over 60 minutes. The resulting solution was heated to 60°C under vigorous stirring and N₂ flow for 3 hours, then stirred overnight at room temperature. Structural characterization by high-resolution transmission electron microscopy showed that the two domains, both with cubic fcc Bravais lattice, can share a common {111}, {100}, or {110} facet, depending on the size of the initial CoPt₃ seeds. Magnetization measurements supported a ferromagnetic CoPt₃ phase relatively low anisotropy due to their disordered crystalline structure. Meanwhile, in Langmuir, Glaser and group synthesized particles with Au and Fe₃O₄ components using controlled nucleation and epitaxial growth of a single iron oxide particle to about 7 nm in diameter onto the surface of a single-crystalline gold seed, about 10 nm in diameter, acting as a precursor. Using pendant drop tensiometry, they found that the amphiphilicity derived from the Janus character of the particles leads to a significantly higher interfacial activity compared to that of the respective homogeneous particles of the same size. The particles could self-assemble at a hexane-water interface, resulting in a significant decrease in the interfacial tension. Furthermore, interfacial activity could be controlled by tuning the particle amphiphilicity, via ligand exchange and coordination of dodecanethiol or octadecanethiol to the gold component.

References:

• Pellegrino, T.; Fiore, A.; Carlino, E.; Giannini, C.; Cozzoli, P. D.; Ciccarella, G.; Respaud, M.; Palmirotta, L.; Cingolani, R., and Manna, L.: Heterodimers Based on CoPt₃-Au Nanocrystals with Tunable Domain Size. J. Amer. Chem. Soc., 128, 6690-6698 (2006).
  • Abstract (courtesy of American Chemical Society Publications).

• Glaser, N.; Adams, D. J.; Boker, A., and Krausch, G.: Janus particles at liquid-liquid interfaces. Langmuir, 22, 5227-5229 (2006).
  • Abstract (courtesy of American Chemical Society Publications).

Graf and group, in Angewandte Chemie International Edition, report a novel catalytic signal amplification method: a DNA probe that, upon hybridization, releases a metal ion which then catalyzes the conversion of a non-fluorescent substrate to a fluorescent product. The probe is a 20-mer oligonucleotide with a 2,2:6,2-terpyridine (tpy) moiety attached to both termini; this chelates a copper (II) ion, as shown by spectrophotometric titration. Upon target binding, chelation is disrupted and the copper (II) ion released. The catalytic module of the nucleic acid detection assay comprises the bidentate chelator and precatalyst 1,10-phenanthroline (phen), its cofactor the released copper(II), the nonfluorescent substrate 2,7-dichlorodihydrofluorescein (DCFH), dioxygen from air as an oxidant, and cysteamine (H₂NCH₂CH₂SH) as a reducing cosubstrate. The mechanism may include the [Cu(phen)]-promoted generation of H₂O₂ from cysteamine and O₂, followed by [Cu(phen)]-catalyzed H₂O₂ oxidation of DCFH to DCF. Although this reaction likely also generates OH radicals or other aggressive oxygen species, no significant cleavage of oligo-DNA is detectable by HPLC under assay conditions, possibly because the DCFH functions as an antioxidant and protects the DNA from degradation. The method was found to be sensitive to single base mismatches: in the presence of a target with a single-base mismatch, the rate of DCF formation was only slightly above background level. Complementary DNA is detectable at 5 x 10^-9 (250 fmol in 1 mL). After 12 hours reaction time, the fluorescence of the probe and target can even be detected by eye.

Reference:

Graf, N.; Goritz, M., and Kramer, R.: A Metal-Ion-Releasing Probe for DNA Detection by Catalytic Signal Amplification. Angew. Chem. Int. Ed. Engl., 45, 4013-4015 (2006).

• Details (courtesy of Wiley Interscience).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .