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N A N O P R O B E S     E - N E W S

Vol. 7, No. 11          November 15, 2006


Updated: November 15, 2006

In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

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Deconstructing Photosystem II with NTA-Ni(II)-Nanogold®, Continued...

Photosystem II has been most extensively probed with NTA-Ni(II)-Nanogold®. Previously, Büchel and co-workers had localized the PsbH subunit in PSII from the green alga Chlamydomonas reinhardtii, and Bumba and group had localized the same subunit in PSII extracted from Synechocystis 6803. This month, Promnares, Bumba and colleagues report in the Journal of Biological Chemistry the successful use of the same method to localize another photosystem II subunit, Small Chlorophyll-binding Protein ScpD (HliB), in Synechocystis 6803, and to establish its spatial relationship with the previously identified PsbH subunit.
NTA-Ni(II)-Nanogold is a novel probe in which, instead of an antibody, the nitrilotriacetic acid (NTA) nickel (II) chelate moiety is linked to gold nanoparticles, and used to target the gold labels to sites marked with engineered polyhistidine tags. This probe has several significant advantages over conventional antibody probes:

  • The nitrilotriacetic acid - Ni(II) chelate is much smaller than an antibody or protein. Labeling resolution is therefore higher: once it has bound, the gold is much closer to its target. This makes NTA-Ni(II)-Nanogold ideal for localizing sites in protein complexes at molecular resolution.

  • Because it is so small, NTA-Ni(II)-Nanogold is also better able to penetrate into specimens and access restricted sites within them.

  • NTA-Ni(II)-Nanogold is prepared using a modified gold particle, with very high solubility and stability. At 1.8 nm in size, is readily visualized by electron microscopy.

  • Binding constants for Ni(II)-NTA are very high due to the chelate effect of multiple histidine binding and multiple Ni(II)-NTA functionalization. Dissociation constants are estimated to be between 10-7 to 10-13 M-1. For many applications, this provides binding strengths comparable to antibodies.

[NTA-Ni(II)-Nanogold, PSII Labeling (54k)]

left: Structure of Ni-NTA-Nanogold® showing interaction with a His-tagged protein. Inset shows localization of labeled subunits within Photosystem II using NTA-Ni(II)-Nanogold. right: Knob protein from adenovirus cloned with 6x-His tag, labeled with Ni-NTA-Nanogold, column purified from excess gold, and viewed in the scanning transmission electron microscope (STEM) unstained (Full width approximately 245 nm).

Cyanobacteria contain several genes coding for small one-helix proteins, called CAB-like proteins (SCPs) or HLIPs, which have significant sequence similarity to chlorophyll a/b-binding proteins. In order to localize one of these proteins, ScpD, in the cells of the cyanobacterium Synechocystis sp. PCC 6803 and to elucidate its function, the authors constructed several mutants of Synechocystis 6803 ScpDHis strain in which ScpD protein is tagged with the His6 epitope (His tag) on its N terminus, expressed under psbA2 promoter. Protein analysis of thylakoid membranes or isolated Photosystem II (PSII) was conducted using two-dimensional Blue native/SDS, two-dimensional native Deriphat/SDS electrophoresis, and immunoblotting using antibodies against raised in rabbits against residues 5886 of the spinach D1 polypeptide, residues 380394 of barley CP47, and monoclonal anti-polyhistidine (Sigma). The results indicated that after high-light treatment, most of the ScpDHis protein in a cell is associated with PSII. The ScpDHis protein was present in both monomeric and dimeric PSII core complexes, and also in the core subcomplex lacking CP43. However, in a mutant lacking the PsbH subunit, the association with PSII was removed. In a PSII mutant lacking cytochrome b559, which does not accumulate PSII, ScpDHis was associated with CP47.

The interaction of ScpDHis with PsbH and CP47 was confirmed by electron microscopy of PSII using NTA-Ni(II)-Nanogold to label the His-tagged protein. Well-washed thylakoid membranes were suspended in 20 mM Tris (pH 7.4) with 100 mM NaCl, and 5 mM CaCl2 at a Chl concentration of 100 µg ml-1, and mixed with an equal volume of NTA-Ni(II)-Nanogold solution (30 nM). The solution was incubated for 30 minutes at 4°C, and free Ni(II)-NTA groups saturated with 1 mM L-histidine for a further 10 minutes on ice. Thylakoid membranes were then solubilized with 1% n-dodecyl-beta-maltoside (DM) for 15 minutes. The unsolubilized material was removed by centrifugation at 60,000 g for 30 minutes and the supernatant loaded onto a freshly prepared 0.11.2 M continuous sucrose density gradient prepared by freezing and thawing the centrifuge tubes filled with 20 mM Tris (pH 7.4), 0.6 M sucrose, 10 mM NaCl, 5 mM CaCl2, and 0.05% DM, and the PSII complexes were separated by centrifugation at 150,000 x g (Hitachi, P56SW rotor) for 14 hours at 4°C. The lower green band containing the PSII dimers was harvested with a syringe and loaded onto a Sephadex G-25 (Amersham Biosciences) desalting column equilibrated with 20 mM Tris (pH 7.4) containing 0.05% DM. Nonlabeled PSII particles were prepared in the same manner, but omitting the NTA-Ni(II)-Nanogold labeling step.

Freshly prepared labeled PSII complexes eluted from the desalting column were placed on glow-discharged carbon-coated copper grids and negatively stained with 0.75% uranyl acetate. Electron microscopy was performed using 80 kV at 60,000 magnification: micrographs free from astigmatism and drift were scanned with a pixel size corresponding to 4.5 Å at the specimen level. Image analysis was then conducted using SPIDER software. About 2900 top-view projections of unlabeled particles and 472 side-view projections of labeled particles were selected from 63 micrographs of the PSII cores for analysis. Both separate data sets were rotationally and translationally aligned, and subjected to multivariate statistical analysis in combination with classification. Classes from each of the subsets were used for refinement of alignments and subsequent classifications. For the final sum, the best of the class members were summed using a cross-correlation coefficient of the alignment procedure as a quality parameter. Image resolution was calculated using the Fourier ring correlation method, and coordinates for molecular modeling were taken from the Protein Data Bank; overlay graphics were generated by the freeware program Accelrys ViewerLite 4.2.

Single particle image analysis identified the location of the labeled ScpDHis at the periphery of the PSII core complex in the vicinity of the PsbH and CP47. Because of the fact that ScpDHis did not form any large structures bound to PSII and because of its accumulation in PSII subcomplexes containing CP47 and PsbH, it is suggested that ScpD is involved in a process of PSII assembly/repair during the turnover of pigment-binding proteins, especially CP47.

Reference:

  • Promnares, K.; Komenda, J.; Bumba, L.; Nebesarova, J.; Vacha, F., and Tichy, M.: Cyanobacterial Small Chlorophyll-binding Protein ScpD (HliB) Is Located on the Periphery of Photosystem II in the Vicinity of PsbH and CP47 Subunits. J. Biol. Chem., 281, 32705-32713 (2006).

More information:

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Nanogold® Labeling Reagents: Better Performance and Updated Instructions

As a result of a comprehensive series of improvements to our manufacturing process, we have been able both to improve the performance of our Nanogold® labeling reagents, Monomaleimido Nanogold and Mono-Sulfo-NHS-Nanogold to offer greater reliability and consistency in their reactivity. Performance enhancements include:

[Nanogold Labeling Reagents and Reactions (37k)]

Monomaleimido Nanogold and Mono-Sulfo-NHS-Nanogold labeling reagents and labeling reactions. Reactivity occurs only through the reactive groups: the remainder of the Nanogold particle is coated with solubilizing ligands and does not adsorb to biological materials.

  • More efficient labeling. You now need to use only a 2-fold to 3-fold excess to achieve efficient labeling of larger biomolecules, so your Nanogold labeling reagents will go further. For example, with one 30 nmol vial of Nanogold, you can now label up to 2.25 mg of IgG, or 0.75 mg of Fab' fragments.

  • Higher solubility and improved solution. These reagents now dissolve more easily and more completely in water. However, should you be labeling a molecule which prefers an organic solvent such as DMSO or isopropanol, Nanogold has high solubility in these solvents, as well as other alcohols, acetonitrile, N,N-dimethylacetamide (DMA) and alcohol-dichloromethane mixtures.

  • Greater thermal stability. This provides added confidence during most high-temperature embedding procedures, and also extends the applications of our reagents and conjugates.

Although thermal stability after conjugation is improved, you should avoid elevated temperatures during labeling, as this will accelerate hydrolysis of the reactive functional groups and decrease your labeling efficiency. In the unlikely event that your reagent is slow to dissolve, you should try vortexing, as this will help to more quickly dissolve any larger buffer crystals and trapped Nanogold reagents. If you are using small volumes or are concerned about the solubility of your reagents during conjugation, add 5 or 10% isopropanol to the reaction mixture; this will assist solution without raising the temperature excessively.

Addition of DMSO to aqueous solutions, or pre-dissolving in DMSO then adding water, should be avoided. Mixing DMSO with water generates heat that may accelerate hydrolysis and reduce your labeling efficiency.

If you are labeling an organic-soluble molecule, such as a peptide or other small organic, we recommend that you first dissolve the Nanogold reagent in DMSO or the aqueous solvent of choice, then add an organic solution of the molecule to be labeled. Gentle stirring overnight is usually required to ensure complete labeling. Note that Nanogold labeling reagents are lyophilized from buffered solutions, in order to ensure that the pH of the reconstituted solutions is optimum for labeling: if you are working in organic solvents, you may notice some residual precipitate. This is simply the residual buffer salt and is not cause for concern, and may be readily filtered off once reaction is complete.

More information:

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NanoVan and Nano-W Help Resolve a Sticky Problem

Negative stains are used to define the edges of particulate or suspended specimens with low contrast, such as protein complexes or viruses, for high-resolution electron microscopy. They are particularly important for structural studies of viruses and other macromolecular protein assemblies with a defined assembly pattern, where visualization of the entire structure and its orientation is required, such as for image analysis, rather than the localization of a specific site by gold labeling.

Negative stains are amorphous, since crystallization can obscure features of interest. When used with ultrastructural gold labeling using smaller gold particles such as Nanogold®, it is helpful if the stain is not too electron-dense, so that contrast between the gold particles and their environment is preserved. Nanoprobes offers two novel negative staining reagents, NanoVan and Nano-W. These are based on vanadium and tungsten respectively, and enable negative staining with a range of different densities. NanoVan is recommended for use with Nanogold because the lower atomic number of vanadium means that the stain is less electron-dense than heavy metal-based stains such as uranyl acetate or lead citrate, and allows easier visualization of the Nanogold particles. It is very fine-grained and highly amorphous, and has been used for a number of high-resolution STEM and TEM studies of virus and protein ultrastructure. Nano-W gives a more dense stain, and is more suited to use with larger gold labels.

Advantages of these reagents:

  • NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give any desired intermediate stain density.
  • Near-neutral pH results in better ultrastructural preservation.
  • NanoVan is less susceptible to electron beam damage than uranyl acetate.
  • Fine grain allows high imaging resolution.

[Negative Staining - Principle and Examples (41k)]

Schematic showing how negative stains work (left) and high-resolution electron micrographs obtained using a scanning transmission electron microscope. (a) Tobacco Mosaic Virus (TMV) negatively stained with 2 % uranyl acetate; (b) TMV stained with 1 % methylamine vanadate (NanoVan); both samples imaged with a dose of 104 eI/nm2. Original full width 128 nm for each image. (c) Side view of groEL (large arrow) labeled with 1.4 nm gold cluster (Nanogold, small arrow) imaged in methylamine vanadate. Note clear visibility of subunit structure and gold cluster. Full width 128 nm. Specimen kindly provided by A. Horwich, Yale University.

Recently, Ruiz and co-workers used a series of different negative staining agents, including NanoVan and Nano-W, uranyl acetate, phosphotungstic acid and ammonium molybdate, to investigate the adhesion of the bacterium Actinobacillus actinomycetemcomitans to collagen. This organism is a gram-negative, facultative, anaerobic bacterium that colonizes the human oral cavity and upper respiratory tract. It is strongly associated with localized aggressive and adult periodontitis, and is the causative agent for other serious systemic infections.

Recently, we the authors identified a protein, EmaA (extracellular matrix protein adhesin A), that mediates the adhesion of A. actinomycetemcomitans to collagen. Because of its conserved sequence and predicted secondary structure, it was believed that EmaA is an orthologue of the Yersinia enterocolitica adhesin YadA. Electron microscopy images of A. actinomycetemcomitans revealed antenna-like protrusions associated with the surface of the bacterium. These structures are absent on emaA mutant strains and can be restored by transformation of the mutant strain with emaA in trans. In order to better evaluate its structure and function, the wild-type and mutant bacteria were examined using high-resolution electron microscopy with negative staining and image processing to characterize these structures in more detail.

For electron microscopy, a small aliquot (5 µl) of the bacterial suspension was diluted in phosphate-buffered saline (PBS) (10 mM sodium phosphate with 150 mM NaCl, pH 7.4), to a concentration providing good coverage on the grid. This was applied to 400-mesh copper grids coated with a thin carbon film. After washing by gently streaming several drops of PBS over the grids, they were negatively stained by running a few drops of negative stain (1% uranyl acetate, 2% phosphotungstic acid adjusted to pH 7 with sodium hydroxide, 2% ammonium molybdate adjusted to pH 7 with ammonium hydroxide, 2% methylamine tungstate (Nano-W), or NanoVan) over them. The last drop was left on the grid for 30 seconds. Excess liquid was wicked off and the grids quickly air dried. Specimens were observed using a Tecnai 12 electron microscope equipped with a LaB6 cathode operated in point mode and a 14-µm 2,048 x 2,048-pixel charge-coupled-device (CCD) camera. The microscope was operated under conditions identical to those used in the past to obtain images that show Thon rings beyond 0.9 nm resolution in vitreous ice preparations. Images were recorded at an accelerating voltage of 100 kV and nominal magnifications in the range of X 40,000 to X 70,000 under low-dose conditions on either film (S0-163; Kodak) or the CCD camera.

For image processing, negatives were scanned with a 7-µm raster size on a flatbed scanner and the images were converted to SPIDER format and reduced by binning two times to a final pixel size on the image of 0.27 nm (nominal magnification, X 52,000). The appendages were boxed from the images using the command "helixboxer" in EMAN. The stalk regions were extracted from these boxes and processed to determine their diameters. In a first analysis, the straightest stalks were selected and subjected to high-pass and low-pass filtrations before the density profile was calculated (the density across the filament axis of the appendage was projected onto the short axis). For more accurate diameter analysis, stalk images were cut into smaller segments (64 by 64 pixels) using "boxer" in EMAN. The new images were aligned to the projection of a model cylinder: rotational alignment was performed using self-correlation functions followed by translational alignment perpendicular to the cylinder axis only. The aligned images were analyzed by a self-organizing map algorithm to check for consistency. All nodes in the resulting output map of the neural network showed centered rods with similar diameters, with the only major differences being the surrounding stain distributions. A rotationally symmetrized three-dimensional reconstruction was calculated from the average of the aligned projections.

The antenna-like structures are composed of a long rod that terminates in an ellipsoidal head region; the head domain can be defined by three different diameters (d): d1 = 2.8 ± 0.3 nm; d2 = 3.1 ± 0.3 nm; and d3 = 4.6 ± 0.3 nm. The stalk has a bend, indicating some degree of flexibility, and the distance from the end of the head domain to the bend in the stalk is highly conserved at 24.6 ± 0.9 nm. Loss of these structures was associated with a decrease in the binding of this bacterium to collagen. The initial estimate of the dimensions of these structures provided by image processing techniques suggests that the appendages are oligomeric structures formed by either three or four subunits. Taken together, the data suggest that emaA is required for the expression of novel appendages on the surface of A. actinomycetemcomitans that mediate the adhesion of the bacterium to collagen.

Reference:

  • Ruiz, T.; Lenox, C.; Radermacher, M., and Mintz, K. P.: Novel surface structures are associated with the adhesion of Actinobacillus actinomycetemcomitans to collagen. Infect. Immun., 74, 6163-6170 (2006).

More information:

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Nanogold® Shows How Skeletal Muscle Burns Fat

Thorkil Ploug is a long-time user of Nanogold® conjugates. In a recent publication in the Journal of Lipid Research with Prats and colleagues, he uses Nanogold-Fab' fragments to help find out how skeletal muscle deals with triglycerides. Nanogold-Fab' conjugates are the smallest commercially available immunogold probe, and consequently achieve both a high degree of penetration - up to 40 µm into cells and tissue sections - and more quantitative labeling than larger probes. In this case, these advantages allow the immunogold confirmation of changes in the distribution and tissue density of a target.

Skeletal muscle lipid metabolism is incompletely understood. In particular, more information is needed to establish the molecular mechanisms that connect intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. It has been extensively debated whether IMTGs are used during exercise, but today this question remains unresolved. Paradoxically, endurance training is well known to increase insulin sensitivity, whereas it has also been shown to increase IMTG content. IMTG is stored within intracellular lipid droplets (LDs), containing a core of cholesteryl esters and triglycerides (TGs) within a phospholipid monolayer surface of unique fatty acid composition containing a complex network of filaments and tubular structures. Several proteins have been reported to associate with LDs, most notably the PAT family proteins perilipin, adipocyte differentiation-related protein (ADRP), and 47 kDa tail-interacting protein (TIP47). Both ADRP and TIP47 show sequence similarity to perilipin, but while perilipin is expressed only surrounding LDs in adipocytes and steroidogenic cells, ADRP is expressed ubiquitously, and TIP47 in some nonadipose cells.

Hormone-sensitive lipase (HSL) is accepted as the lipolysis rate-limiting enzyme in adipocytes, but an increasing body of evidence suggests that other neutral lipases are active in skeletal muscle. However, recent findings that HSL has 10-fold higher activity toward diacylglycerol than toward TG, and that lipolysis in HSL-knockout mice causes diacylglycerol accumulation, indicate that HSL is primarily responsible for diacylglycerol hydrolysis rather than initial TG hydrolysis. A recently discovered triglyceride lipase, adipose triglyceride lipase, has been found to correspond to the distribution of HSL-knockout mouse diacylglycerol accumulation, and it has been suggested that the hydrolysis of the first ester bond in TG is catalyzed predominantly by adipose triglyceride lipase. Epinephrine and muscle contraction increase neutral lipase activity in rat skeletal muscle homogenate, but this can be blocked with anti-HSL antibodies, indicating that HSL participates in the rate determining step. In human skeletal muscle, total neutral lipase activation is reported during exercise, ascribed to HSL: this confirms that HSL is the exercise-induced lipolysis rate-limiting enzyme.

However, lack of correlation between HSL activation measured chemically in vitro and lipolysis rates has been reported frequently. One explanation is HSL intracellular redistribution, in which HSL is targeted to the substrate in response to lipolytic stimuli: this mechanism that cannot be detected in vitro because of disruption of cellular integrity. Translocation of HSL to LDs has been demonstrated in adipocytes in response to lipolytic stimuli, but is unable to do so in the absence of perilipin. Mutation of the N-terminal cyclic AMP-dependent protein kinase (PKA) sites of perilipin abolishes PKA-induced lipolysis. This supports the hypothesis that perilipin forms a barrier around LDs and that phosphorylation of perilipin by PKA induces a conformational change providing HSL access to TG. Given the importance of HSL intracellular distribution and perilipin in lipid metabolism regulation in adipocytes, the authors conducted the current study to determine whether a similar process occurs in nonadipose skeletal muscle cells, which do not express perilipin, and if so, which proteins may be involved.

The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Confocal and transmission electron microscopy were used to analyze the intracellular distribution of ADRP, TIP47, and HSL in relation to intramuscular triglycerides in skeletal muscle at rest and after stimulation with epinephrine or muscle contraction. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 minutes, and single muscle fibers used for morphological analysis by confocal and transmission electron microscopy. A decrease in IMTG was found in response to both lipolytic stimuli. Using immunofluorescence, two PAT family proteins, ADRP and TIP47, were found to be associated with IMTG.

Both immunofluorescence and electron microscopy were used to study HSL distribution. In preparation, bundles of 13 individual fibers were teased from fixed muscles with fine forceps, and transferred to 50 mM glycine in PBS. Nonspecific binding was blocked with a solution of 50 mM glycine, 0.25% bovine serum albumin, 0.03% saponin, and 0.05% sodium azide in PBS for 30 min. Fibers were then incubated overnight with rabbit anti-HSL primary antibody raised and affinity-purified against recombinant rat HSL, diluted in blocking buffer supplemented with 200 µg/ml goat IgG. After washing (3 X 30 minutes) they were incubated for 2 hours with Nanogold-Fab' goat antirabbit fragments diluted 1:300 in blocking buffer. After washing in blocking buffer followed by PBS, the fibers were fixed for 1 hour at room temperature in 2.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. After several washes in water, the specimens were enhanced with HQ Silver for 6 minutes, then washed in water and stored overnight in 0.1 M phosphate buffer at 4°C. Fibers were then treated with 0.5% osmium tetroxide in 0.1 M phosphate buffer for 20 minutes, en block mordanted for 1520 minutes in 2% uranyl acetate in 50% acetone, dehydrated through a graded series of acetone, infiltrated with epoxy resin Polybed 812, horizontally embedded in flat molds, and cured for 2 - 3 days at 55°C. Usually, sections were poststained with aqueous lead citrate for 5 minutes. Samples were examined in a Philips CM 100 transmission electron microscope operated at an accelerating voltage of 80 kV.

In both basal and stimulated muscle fibers, a variable fraction of HSL was associated with LDs, while the remainder was found in the cytoplasm as small clusters. After either epinephrine stimulation or muscle contraction, an increase in HSL associated with LDs was detected. These results confirm HSL translocation to the LDs in response to stimulation both with epinephrine or contraction, and confirm that HSL distribution plays a role in lipid metabolism regulation in skeletal muscle cells.

Reference:

  • Prats C.; Donsmark M.; Qvortrup K.; Londos C.; Sztalryd C.; Holm C.; Galbo H., and Ploug T.: Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine. J. Lipid Res., 47, 2392-2399 (2006).

Reference - detailed Nanogold and silver enhancement procedure:

  • Ploug, T.; van Deurs, B.; Ai, H.; Cushman, S. W., and Ralston, E.: Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions. J. Cell. Biol., 142, 1429-46 (1998).

More information:

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Changes to Newsletter Sign-up and Security

Recently, we have experienced a large number of attempts to abuse the forms-to-email script on our web site to try to send spam through our servers. Our servers are protected from this type of attack, but in order to ensure the highest level of protection against future developments, and to make sure that all requests are reviewed for legitimacy before acceptance, we have modified the sign-up and unsubscribe procedures slightly. When you complete the form, the results no longer go directly to the mailing list processing software, but to us, where they are checked and entered manually. This may result in a few hours delay in signing you up if you fill out the form outside our office hours, but this gives the best possible security for our subscribers and for ourselves.

Nanoprobes does not share information from our newsletter sign-up with any other organizations or third parties: we use the information to help us make the newsletter more responsive to your interests, and to better understand our customers so that we can develop better products and services. If you have any questions or concerns about this newsletter, or want to give us feedback, please e-mail us.

New on our web site: more on the development of gold nanoparticles as X-ray contrast agents. See our press release for details. See all our past news stories and press releases on our News index page.

More information:

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Other Recent Publications

Bottom-up spatial organization of potential nanoelectronic components is a key intermediate step in the development of molecular electronics, and Zheng and co-workers advanced the field further with their recent paper in Nano Letters describing the use of rigid, self-assembling triangular DNA motifs for periodic ordering of 5 and 10 nm gold particles in two dimensions. The authors describe robust three-space-spanning DNA motifs that are used to organize nanoparticles in two dimensions. One strand of the motif ends in a gold nanoparticle; only one DNA strand is attached to the particle. By using two of the directions of the motif to produce a two-dimensional crystalline array, one direction is free to bind gold nanoparticles. Identical motifs, tailed in different sticky ends, enable the two-dimensional periodic ordering of 5 and 10 nm diameter gold nanoparticles. Several motifs have been built that span three-space, for example six-helix bundles. One of these motifs, termed a 3DDX triangle, contains double helix molecules in each of its three domains. It is possible to produce 2D lattices with this motif if only two of the linearly independent directions contain cohesive ends. This leaves a third direction not involved in lattice formation, and its blunt end can be used as a site to include a gold nanoparticle. Specificity is increased by using a nanoparticle that contains only a single DNA strand; this strand is one of those that form the motif and its 5' end is on one of the blunt ends. These features provide for a system that combines all of the known robustness features for this system. 5 or 10 nm gold particles are attached to the triangles following two-step electrophoretic isolation processes. In the first step, thiolated single-stranded DNA (ssDNA) is reacted directly with 5 or 10 nm gold nanoparticles. Discrete bands of low mobility that appear in the same lane (Figure S2) on an agarose gel correspond to a defined number of strands per particle: the band corresponding to nanoparticles with one ssDNA was isolated from the other bands and recovered. The highly purified DNA-gold conjugates were added to the solution containing all the other component strands, to form the 3D DX triangle. Following a similar isolation procedure, the collected 3D DX triangle-Au conjugates were mixed with the complementary 3D DX triangles, to form a two-triangle array. In this manner, arrays were formed in which 5 nm particles were alternated with unlabeled triangles, complementary 5 nm gold-decorated triangles were assembled, and 5 nm gold particles alternated with 10 nm ones. Atomic force microscopy and transmission electron microscopy indicated that the particle spacing was highly regular in all cases.

Reference:

  • Zheng, J.; Constantinou, P. E.; Micheel, C.; Alivisatos, A. P.; Kiehl, R. A., and Seeman, N. C.: Two-dimensional nanoparticle arrays show the organizational power of robust DNA motifs. Nano Lett., 6, 1502-1504 (2006).

Mayer and group report the expansion of the functionalization chemistry of ligand-stabilized gold nanoparticles to include polypyridyl ruthenium complexes in their recent paper in Chemical Communications. Two heteroditopic ligands were synthesized, (4-(2-mercaptopyridyl)imidazo[4,5-f])-2,9-dimethyl-1,10-phenanthroline (NeoSH) and 4'-(2-mercaptopyridyl)-2,2':6',2''-terpyridine (TerSH): both possess the 2-mercaptopyridine (2-Mpy) group to ensure the connection with the metallic gold nanoparticle surface, and either a bidentate (NeoSH) or a tridentate (TerSH) pendant group used to generate the ruthenium complex. Both ligands were used to stabilize and functionalize gold nanoparticles. Coordinated gold nanoparticles were prepared by reduction of tetrachloroauric acid in the presence of the ligands. Stable, reproducible and monodisperse gold nanoparticles with a diameter of 3.8 ± 0.6 nm were characterized in DMF, without formation of aggregates, by transmission electron microscopy. These functionalized gold nanoparticles have been used as effective platforms to functionalize [Au]-NPs by polypyridyl ruthenium complexes. In order to generate the ruthenium complexes, TolylTerpyRuCl3 (TolylTerpy: 4'-(4-tolyl)-2,2':6',2''-terpyridine) was reacted with [Au]-TerSH in a water/ethanol (v/v, 1 : 5) at 80°C, and followed by UV-vis spectroscopy over a period of 16 hours. These studies showed that the complexation of TolylTerpyRuCl3 with [Au]-TerSH-NCs proceeds in two steps: TolylTerpyRuCl3 is first solubilized, and then reacts with [Au]-TerSH-NCs. The formation of the ruthenium complex [TolylTerpyRuTerSH]2+ was confirmed by the decrease of the band at ca. 385 nm and concomitant increase of the bands at 280, 315 and 491 nm, characteristics of [TolylTerpyRuTerSH]2+ (Fig. 2). TEM analysis (Fig. 3) showed that the size, shape and dispersity of the [Au]-NCs were not significantly affected by ruthenium complex formation, which confirms the high stability of the [Au]-TerSH-NCs even in refluxing water-ethanol mixtures. Similar reactivity and stability were observed during the reaction of complexation of Au-NeoSH-NCs with (Phen)2RuCl2.

Reference:

To conclude a triple-play of novel nanotechnology publications, Biju et al report, in a recent issue of the Journal of Photochemistry and Photobiology A: Chemistry, the preparation and photoluminescence characteristics of carbon nanotubes decorated with both gold particles and semiconductor nanoparticles or "quantum dots." Two types of nanoscale hybrid materials were synthesized by conjugating CdSeZnS quantum dots (QDs) and gold nanoparticles (NPs) to sidewall functionalized single-walled carbon nanotube (SWNT) templates, For sidewall functionalization of SWNT, an addition reaction with p-nitrobenzenediazonium tetrafluoroborate was used; the nitro-SWNTs were separated by solvent extraction and silica gel filtration, then reduced to amino-derivatives for conjugation. The amino-SWNT was conjugated to goldNPs and QDs through ammonium salt formation using mercaptoacetic acid-functionalized gold nanoparticles, and biotinylation using NHS-biotin followed by reaction with quantum dot-streptavidin, respectively. Photoluminescence (PL) properties of the hybrid materials, SWNTQD and SWNTgoldNP conjugates, were then investigated. Excessive sidewall functionalization of SWNTs into nitro- and amino-derivatives provided weak PL and water solubility to the SWNT derivatives. Solubility of SWNT derivatives in aqueous media proved helpful for efficient conjugation of SWNTs to quantum dots and gold nanoparticles. The SWNTQD and SWNTgoldNP conjugates were characterized using atomic force microscopy (AFM) imaging. AFM imaging confirmed that the sidewall functionalized SWNTs assisted the formation of one-dimensional close-conjugates of QDs and goldNPs. Although the nitro- and amino-derivatives of SWNT showed weak PL, conjugation of goldNPs to the amino-derivative quenched the PL quantitatively. On the other hand, conjugation of luminescent QDs to SWNTs resulted in a partial (~35%) quenching of PL from QDs. The quenching of PL from SWNTs by goldNPs was attributed to non-radiative energy transfer between SWNTs and goldNPs or vice versa; the quenching of PL from QDs was attributed to non-radiative energy transfer from QDs to SWNTs. In the current work, a simple method of close-packing of QDs and goldNPs using functionalized SWNT templates was demonstrated, and it was confirmed that photoluminescence properties of SWNTs and QDs are affected in the close-packed structures. SWNT and NP-based hybrid materials show great promise as building blocks for nanoscale devices, and the current study provides potentially useful information for the design of various hybrid nanoscale materials for device applications.

Reference:

  • Biju, V.; Itoh, T.; Makita, Y., and Ishikawa, M.: Close-conjugation of quantum dots and gold nanoparticles to sidewall functionalized single-walled carbon nanotube templates. J. Photochem. Photobiol. A, Chem., 183, 315-321 (2006).

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