Updated: April 12, 2004
N A N O P R O B E S E - N E W S
Vol. 5, No. 4 April 12, 2004
In this Issue:
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This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.
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To make the e-mail edition of our newsletter quicker and easier to browse, it now includes only a brief introduction to each article. This links to the complete article hosted here on our web site, with references, links, improved graphics and better navigation. We hope that you will find our new format easier to use; as always, let us know if we can improve anything.
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Gold particles are key players in nanobiotechnology the convergence of nanotechnology with biological design. The wide variety of optical and electronic properties of gold clusters and nanoparticles give them many potential nanotechnology applications. Gold clusters with chemically selective reactivity possess another important advantage: they may be conjugated to specific sites within biological molecules where these properties impart useful functionality. This field is described in a new book, Nanobiotechnology: Concepts, Applications and Perspectives," edited by Mirkin and Niemeyer, which contains several chapters on the applications of gold particles, including contributions from ourselves and our collaborators that describe many applications of our Nanogold® labeling technology.
"Nanobiotechnology: Concepts, Applications and Perspectives" - details and ordering information
Meanwhile, LaBean and co-workers have described the use of triple-crossover DNA tiles, modified with thiols, to form self-assembled DNA nanotubes that are then metallated and used to prepare conductive nanowires. The structural motifs were constructed using oligoucleotides about 50 bases in length, previously shown to form the desired structures, but modified with thiols in such a way that the thiol-bearing portion projected out of the plane of the tile. These were reduced with dithiothreitol (DTT) before use and exchanged into annealing buffer using gel permeation over a NAP5 column. Oligonucleotides were annealed to form tiles and lattices in stoichiometric mixtures by heating to 95°C for 5 minutes, then slowly cooling to room temperature in a thermal cycler by dropping 0.1°C per minute (total annealing time close to 12 hours) to produce a nanotube lattice.
This lattice was then metallated using a two-step procedure, previously reported by Yan and co-workers, but modified so that the initial silver seeding step was carried out in solution rather than on substrate. First, the TX nanotube lattice was seeded with silver using the glutaraldehyde method: annealed DNA was incubated with 0.2 % glutaraldehyde in 1 x TAE / Mg2+ buffer (40 mM Tris acetate, pH 7.6 / 2 mM EDTA / 10 mM MgCl2) on ice for 20 min, then at room temperature for 20 min. The sample was then dialyzed overnight at 4°C in 1 liter of 1 x TAE / Mg2+ buffer. Aldehyde-derivatized DNA was incubated in the dark with an equal volume of 0.1 M AgNO3 in 25% ammonia buffer (pH 10.5) at room temperature for 3090 min, then 10 microliters was deposited onto an aminopropylsilane-treated silicon substrate and allowed to absorb for 5 minutes. Excess reagent was rinsed off with distilled water and the sample was dried under a stream of nitrogen. The second step in the procedure is silver enhancement using HQ Silver for 5 minutes. Finally, excess reagent was rinsed off again with distilled water and the sample dried again under a stream of nitrogen.
Site-selective gold labeling with Nanogold reagents was then used to confirm the locations of specific sites within the nanotubes. Treatment of the tubes with Monomaleimido Nanogold or with colloidal gold did not result in labeling: this indicates that the thiol-bearing stem projects inwards, and confirmed that curvature induced by disulfide formation produces the nanotube morphology (annealing in the presence of 20 mM DTT produced stacked sheets rather than nanotubes). Colloidal gold binding to the ends of the tubes suggested that thiols are exposed here. When aliphatic amines were introduced into the tiles at sites known to project from face opposite to that from which the thiols project, these were successfully labeled with Mono-Sulfo-NHS-Nanogold, confirming the location of the thiols on the inner tube surface.
AFM and SEM indicated that metallation produced nanowires about 40 nm in diameter, 35 nm in height, and up to 5 micrometers in length. Chromium-gold electrodes were patterned onto the wires by electron-beam lithography and used for conductivity measurements: the nanowires were found to be less conductive than bulk silver, possibly due to low-density silver deposition within tubes, but more conductive than some other nanowires produced recently.
Reference:
Liu, D.; Park, S. H.; Reif, J. H, and LaBean, T. H.: DNA nanotubes self-assembled from triple-crossover tiles as templates for conductive nanowires. Proc. Natl. Acad. Sci. USA, 101, 717-722 (2004).
Silver metallation procedure:
Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H.: DNA-templated self-assembly of protein arrays and highly conductive nanowires. Science, 301, 1882-1884 (2003).
More information:
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Because Nanogold® is coated with small organic molecules rather than large proteins or other macromolecules, and each gold particle is linked to only one Fab fragment or IgG molecule, Nanogold conjugates are very similar in size to the unconjugated Fab or IgG molecules. Therefore their ability to penetrate into cells and tissues and access hindered or restricted antigens is often superior to conventional colloidal gold probes; the unique surface functionalization of Nanogold also protects it better against non-specific interactions with other cell and tissue components, so background is lower than with conventional colloidal gold.
Eilbracht, Schmidt-Zachmann and group recently demonstrated this when they used Nanogold to determine the nuclear distribution of NO66, a novel 66 kDa protein that is a highly conserved component of the nucleolus in most cell types. Immunofluorescence and immunogold studies were used to localize NO66 and to determine its degree of colocalization with Ki-67, HP1 alpha, and PCNA in a variety of cultured cell lines. For immunoelectron microscopy, cells grown on coverslips were fixed in 2% formaldehyde in PBS, or methanol (5 min, -20°C) and acetone (30 s, -20°C). For some experiments, cells were incubated with 5 micrograms/ml actinomycin D (AMD) for 4 h before fixation. Incubation with the primary antibody (monoclonal anti-NO66 antibodies, diluted 1:10 or 1:25) was performed in a wet chamber for 5 h at room temperature. After three washes with PBS for 5 min each, bound antibodies were reacted overnight at room temperature with Nanogold-IgG anti-guinea pig immunoglobulin G (IgG), diluted 1:50. After several washes (PBS, 5 minutes each), the cells were fixed with 2.5% glutaraldehyde in 0.05 M cacodylate buffer for 15 min at 4°C. The signals were silver-enhanced using HQ silver for 10 to 15 min at 18°C in the dark, followed by three 5-minute washes with a neutral fixer (20 mM HEPES / 250 mM sodium thiosulfate, pH 5.8). Subsequently, cells were postfixed with 0.01% osmium tetroxide solution and processed for flat embedding in Epon.
Immunolocalization studies found a dual localization pattern for NO66 comprising a strong enrichment in the granular part of nucleoli and in distinct nucleoplasmic entities. Colocalization studies with Ki-67, HP1 alpha, and PCNA showed that the staining pattern of NO66 overlaps with some clusters of late replicating chromatin. Combined with the finding that NO66 cofractionates with large preribosomal particles but is absent from cytoplasmic ribosomes, this led the authors to propose that NO66 participates in the replication or remodeling of certain heterochromatic regions in addition to its role in ribosome biogenesis.
References:
Eilbracht, J.; Reichenzeller, M.; Hergt, M.; Schnolzer, M.; Heid, H.; Stohr, M.; Franke, W. W., and Schmidt-Zachmann, M. S.: NO66, a Highly Conserved Dual Location Protein in the Nucleolus and in a Special Type of Synchronously Replicating Chromatin. Mol. Biol. Cell., 15, 1816-1832 (2004).
Nanogold labeling procedure:
Zirwes, R. F.; Eilbracht, J.; Kneissel, S., and Schmidt-Zachmann, M. S.: A novel helicase-type protein in the nucleolus: protein NOH61. Mol. Biol. Cell, 11, 1153-1167 (2000).
Silver enhancement procedure:
Uchida, N.; Honjo, Y.; Johnson, K. R.; Wheelock, M. J., and Takeichi, M.: The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones. J. Cell. Biol., 135, 767-779 (1996).
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We are sometimes asked whether our silver and gold enhancement reagents or negative stains will work with products from other companies. Since the chemistry is the same, the answer is almost always yes. Fletcher, Smith and co-workers recently provided a good illustration when they used our vanadium-based negative stain, NanoVan, in conjunction with 12 nm colloidal gold probes from another source. The group was investigating the Neisseria meningitides 2086 lipoprotein as a potential vaccine against these bacteria, and used immunogold to study the specificity and strength of binding of antibodies against this target to cell surfaces.
N. meningitides serogroup B strain H44/76 cells were fixed for 60 min at room temperature in 4% paraformaldehyde plus 0.05% glutaraldehyde in PBS, pH 7.2. Droplets of cells were placed on Parafilm and Formvar carbon-coated gold grids were placed face down on each droplet. Excess fluid was wicked off, and blocking was accomplished in two stages using phosphate-buffered saline (PBS) containing 1% bovine serum albumin (PBS-BSA) for 5 min, followed by PBS containing 1% cold water fish gelatin for 10 min. Excess aldehyde was quenched with 0.02 M glycine in PBS for 5 min. Whole-cell, negative-stain, immunogold labeling was performed by inverting cells over rLP2086 antibody derived from strain 8529 diluted 1:50 in PBS-BSA for 1 h in a humidified chamber, rinsing 5 x 1 minute in PBS-BSA, then incubating for 60 min with 12 nm gold-conjugated goat anti-mouse immunoglobulin G plus immunoglobulin M (Jackson ImmunoResearch Labs) diluted 1:5 in PBS-BSA. After rinsing in PBS (4 x 1 minute), grids with cells were stabilized with 1% glutaraldehyde in PBS (3 minutes), rinsed in distilled water (5 x 1 minute). Grids were then negatively stained for 30 seconds using NanoVan.
Reference:
Fletcher, L. D.; Bernfield, L.; Barniak, V.; Farley, J. E.; Howell, A.; Knauf, M.; Ooi, P.; Smith, R. P.; Weise, P.; Wetherell, M.; Xie, X.; Zagursky, R.; Zhang, Y., and Zlotnick, G. W.: Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect. Immun., 72, 2088-2100 (2004).
Unlike 'positive' stains, which stain regions of specimens, negative stains fill in the gaps between features and contrast the edges of particulate or suspended specimens such as protein complexes or cells. Nanoprobes offers two negative stain reagents with complementary properties. NanoVan is recommended for use with Nanogold® because it is based on vanadium and is therefore less electron-dense than heavy metal based stains such as uranyl acetate or lead citrate. Nano-W is based on the heavier element tungsten and therefore gives a more dense stain, and is more suited to use with larger gold labels.
Advantages of these reagents:
- Completely miscible: may be mixed in different proportions to give desired intermediate stain density.
- Near-neutral pH
- NanoVan is less susceptible to electron beam damage than uranyl acetate.
- Fine grain allows high imaging resolution.
For a general introduction to negative staining and its advantages, see Linda Stannards virus ultrastructure pages from the Department of Medical Microbiology at the University of Cape Town.
More information:
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The use of small gold nanoparticles to immobilize proteins for AFM observation has been proposed by Collins et al. Size-selected gold clusters, formulated as Au55+ and Au70+, were pinned to a graphite surface, and the atomic force microscope (AFM) used to visualize two proteins, histidine affinity tagged (nitrilotriacetic acid, or NTA-Ni(II)-modified) green fluorescent protein (pHAT-GFP) and Human Oncostatin M, both in air, and in physiological buffer solution, which mimics their natural environment; this approach enable the visualization of conformation changes in single protein molecules without the distortions induced by crystal packing.
Gold clusters were produced in a magnetron sputtering, gas aggregation cluster beam source and size-selected to within 5% accuracy using a lateral time of flight mass filter. A radio frequency (RF) plasma is ignited in an argon/helium gaseous mixture and held in the vicinity of a gold target by a magnetic field. Sputtered hot gold atoms are subsequently cooled by the helium, resulting in metal cluster formation. Ionization of some gold atoms in the plasma forms a proportion of ionized clusters AuN+, which are accelerated and focused into an ionized cluster beam via a series of electrostatic lenses, size-selected using a time of flight mass selector, and deposited onto graphite substrates at normal incidence and room temperature in vacuum. The deposited clusters were used to bind proteins from solution. In both cases, AFM studies indicated that small islands containing several proteins were deposited.
A strong and selective interaction between the nitrogen atoms of the NTA group and the gold surface was not demonstrated for pHAT-GFP: deposition was made from relatively concentrated solution, suggesting a deposition pattern that was not significantly different from that produced by initiation of precipitation. However, in the case of Human Oncostatin M, which contains both a free thiol and a disulfide, very harsh conditions were required to remove the deposited protein (0.5 M nitric acid): the strength of the attachment indicated by this finding suggests that at least the initial protein chemisorbs to the gold via the strong affinity of thiols for gold. The authors report that GroEL, which contains thiols, and horseradish peroxidase, which contains disulfides, may be immobilized in the same way.
Reference:
Collins, J. A.; Xirouchaki, C.; Palmer, R. E.; Heath, J. K., and Jones, C. H.: Clusters for biology: immobilization of proteins by size-selected metal clusters. App. Surf. Sci., 226, 197-108 (2004).
Nanogold, our 1.4 nm gold cluster, is supplied with a variety of different reactivities based both on selective chemical reactions (such as maleimides for reaction with thiols, and sulfo-NHS-groups for reacting with amines) and on charge (positively and negatively charged Nanogold), and therefore has the potential both to selectively bind to surfaces and to selectively immobilize proteins or other targets with the appropriate reactivity. This also includes the new NTA-Ni(II)-Nanogold, which possesses multiple NTA-Ni(II) chelate groups and binds strongly and selectively to polyhistidine-tagged proteins. In addition, although we do not manufacture conventional colloidal gold, we maintain a technical help section on our web site for those wishing to use this technology.
More information:
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Use of the pre-embedding Nanogold labeling method with HQ Silver enhancement in neuroscience was further extended by Papp and co-workers, who used it to localize HCN2, one of a class of neuroproteins called hyperpolarization-activated cyclic nucleotide-gated cation channel proteins (HCN1-4), which are potentially able to modulate membrane excitability, in the rat spinal dorsal horn. It was found that HCN2 is largely confined to axon terminals with dense-core vesicles, in the central terminals of peptidergic primary afferents. Within these terminals, some of the silver grains marking the accurate location of HCN2 molecules were associated with the cell membrane, almost exclusively in extrasynaptic locations, and others were scattered in the axoplasm. HCN2 may therefore contribute to the modulation of membrane excitability of nociceptive primary afferent terminals in the spinal dorsal horn.
Reference:
Antal, M.; Papp, I.; Bahaerguli, N.; Veress, G., and Vereb, G.: Expression of hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 in axon terminals of peptidergic nociceptive primary sensory neurons in the superficial spinal dorsal horn of rats. Eur. J. Neurosci., 19, 1336-1342 (2004).
Ji et al. demonstrate the sensitivity of silver-enhanced gold for another application: colorimetric detection of gene methylation using a DNA hybridization array coupled with linker PCR (similar to the method of Alexandre). Hybridized slides were incubated for 45 minutes with streptavidingold conjugate diluted 10-fold, washed (5 x 1 minute) with a 10 mM maleate buffer containing 15 mM NaCl and 0.1% Tween, pH 7.5 and incubated at room temperature for 10 min in Silver Blue" solution (the combination in 1:1 (v/v) ratio of two silver salt (AgNO3) and hydroquinone solutions). The slides were rinsed in water, air-dried for 5 hours at 37°C, and read with a fluorescence microscope equipped with a digital camera. Sensitivity was comparable to fluorescence detection using a confocal scanner, with a detection limit of 0.1 fmol of biotinylated target DNA. Other advantages include the stability of the silver deposit, enabling long-term storage of the microarray, and its suitability for use with plastic supports, such as acrylic layer or polycarbonate, which can have strong autofluorescence. We would, however, be grateful if the authors would refrain from calling the gold they used 'Nanogold:' it was actually conventional colloidal gold.
Reference:
Ji, M.; Hou, P.; Li, S.; He, N., and Lu, Z.: Colorimetric silver detection of methylation using DNA microarray coupled with linker-PCR. Clin. Chim. Acta, 342, 145-153 (2004).
Autometallography was also used this time without gold labeling to study the distribution of chelatable zinc in the developing mouse. Nitzan and co-workers used both selenite autometallography and detection with a quinoline-based histofluorescence agent, N-(6-Methoxy-8-quinolyl)-p-Toluene-Sulfonamide (TSQ). Although the staining patterns with both methods were very similar, some differences were observed. The liver was heavily labeled with the autometallography (AMG) method in the late prenatal and neonatal mouse but exhibited virtually no fluorescence with TSQ, possibly because selenite AMG traces zinc indirectly while TSQ is a specific zinc-sensitive fluorophore, and possibly because of their differing chemical properties - Furthermore, selenite and TSQ are inherently different types of molecules, the former selenite is ionic and TSQ hydrophobic. The AMG reaction noted in the liver may also be due to zinc that is dynamically competed for by selenite from the pool of protein-bound zinc.
Reference:
Nitzan, Y. B.; Sekler, I., and Silverman, W. F.: Histochemical and histofluorescence tracing of chelatable zinc in the developing mouse. J. Histochem. Cytochem., 52, 529-539 (2004).
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