Updated: October 6, 2003

N A N O P R O B E S     E - N E W S

Vol. 4, No. 10          October 6, 2003

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.

Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing tech@nanoprobes.com.

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Silver Nanowires Templated by DNA

We have previously described the preparation of DNA strands decorated with positively charged Nanogold®, and suggested in our abstract at Microscopy & Microanalysis '01 that these may form the basis for autometallographically generated molecular wires; since current lithographic chip production techniques produce wires about 0.3 microns in diameter, molecular wires could offer large increases in packing density.

Yan, LaBean and co-workers have reported proof of principle for this approach in their recent paper, in which they also illustrate the power of DNA for both self-assembly and templating other processes. They designed and prepared a DNA nanostructure consisting of four four-arm junctions oriented with a square aspect ratio. Programmable self-assembly of 4 x 4 tiles produced two distinct lattice morphologies, both containing periodic square cavities about 17 nm in size: uniform-width nanoribbons, and two-dimensional nanogrids. The square cavity is bordered by four different tiles, a strategy which allows for the programmed generation of cavities with many different properties. Incorporation of biotin at a T4 loop at the center of the motif and treatment of the nanogrid lattice with streptavidin resulted in the formation of a periodic streptavidin array, which was imaged by AFM.

The 4 x 4 nanoribbons were metallated using a two-step process. First, the annealed ribbon lattice was incubated with 0.2% glutaraldehyde in 1 x tris-acetate-EDTA (TAE)-Mg buffer on ice for 20 minutes then at room temperature for 20 minutes, then dialysed overnight at 4°C in 1 L of 1 x TAE-Mg buffer. The aldehyde-derivatized DNA was then seeded by incubation in the dark with a 0.1 M solution of silver nitrate in 25% ammonia buffer (pH 10.5) at room temperature for 3090 minutes, 10 microliters was deposited onto silicon substrate, and excess reagent was rinsed off with distilled water. In the second step, the deposited DNA was developed using HQ Silver for 5 minutes. In this way nanowires up to 5 micrometers in length, 43 nm in width and 35 nm in height were generated. Conductivity measurements indicated a bulk resistivity of 2.4 x 10-6 ohm-m for the silver nanowires, considerably lower than previously reported values for DNA-templated silver deposition.

Reference:

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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Nanogold® Reveals Function of Eukaryotic RNA Helicase

Although genetic manipulations have revealed the functions of RNA helicases in ribosomal RNA (rRNA) biogenesis in yeast, no report has shown the role of an RNA helicase in rRNA formation in higher eukaryotes. Now, Yang, Ochs and group report the functional characterization of the frog homologue of nucleolar RNA helicase II/Gu (xGu, or DDX21). Down-regulation of xGu in Xenopus laevis oocyte results in the depletion of 18 and 28S rRNAs and an accumulation of 20S, and was also associated with degradation of 28S rRNA into fragments smaller than 18S. These effects are reversed in the presence of in vitro synthesized wild type xGu mRNA, but not a helicase-deficient mutant form, and similar effects resulted from microinjection of anti-xGu antibody.

Pre-embedding Nanogold® labeling with silver enhancement was used to localize this protein. PtK2 cells (rat kangaroo kidney epithelial cells; ATCC), grown as monolayers, were fixed for 30 minutes at room temperature (RT) with 3% paraformaldehyde buffered with PBS, rinsed with PBS, and permeabilized at -20°C in 100% acetone for 1 minute. Following a PBS rinse, cells were blocked for 30 minutes at RT with 1% normal goat serum in PBS (NGS/PBS) and incubated overnight at 4°C with a 1:1000 dilution of human serum containing anti-Gu antibody. Following three 10-minute rinses in PBS and blocking for 30 minutes in NGS/PBS, cells were incubated for 1 hour at RT with shaking in 1.4 nm Nanogold-Fab' anti-human IgG or anti-rabbit IgG diluted 1/100 in NGS/PBS, rinsed in PBS, fixed for 30 minutes in 1% glutaraldehyde/ PBS, rinsed in PBS, rinsed well in distilled water, then silver enhanced for 4 minutes in the dark at RT with HQ Silver, rinsed with water, osmicated for 30 minutes with 1% osmium tetroxide in water, rinsed with water, dehydrated with ethanol, and embedded in Polybed 812. Thin sections were examined unstained.

xGu was found to be localized to the granular and dense fibrillar components of PtK2 cell nucleoli, supporting its involvement in the processing of rRNA. These results show that xGu is involved in the processing of 20 to 18 S rRNA, and contributes to the stability of 28S rRNA in Xenopus oocytes.

References:

More information:

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Gold Nanoparticles in Cell Imaging and Cancer Therapy

Pitsillides and co-workers report a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles, targeted by monoclonal antibodies, which are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates such as singlet oxygen. The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. Strong particle size dependence was found for these interactions.

CD8+ lymphocytes were labeled either with R-phycoerythrin-labeled monoclonal anti-human CD8 mouse IgG followed by 30 nm gold-conjugated anti-mouse IgG, or with biotinylated monoclonal anti-human CD8 mouse IgG bound with 0.83 micron streptavidin-coated iron oxide nanoparticles, added to mixed human lymphocytes together with R-phycoerythrin-labeled monoclonal anti-human CD8 mouse IgG; irradiation at 532 nm was conducted after 30 minutes in cell culture medium, and viability subsequently measured using calcein-AM. The degree of membrane permeabilization was investigated using CD45+ T-lymphocytes, incubated with monoclonal anti-human CD45 mouse IgG followed by 20 nm or 30 nm gold-conjugated anti-mouse IgG; permeability was determined by subsequent treatment with a 10,000 ME fluorescein-conjugated dextran, and cell viability using propidium iodide. With the iron oxide particles, rapid vaporization of buffer in a thin layer around each particle and cavitation bubble formation was visualized using time-resolved light microscopy with stroboscopic illumination. Up to 80 % lethality was found (only 2 % of CD8- cells were killed). With the smaller gold particles, similar events were not visualized, but a lethality of 95 % was found at a count of 500 particles per cell (5 - 8 % for CD8- cells); it was thought that the strong absorbance of colloidal gold at 520 nm helped maximize energy absorption. By modifying conditions, transient membrane permeabilization could be achieved without cell death, and selective inactivation of proteins bound to the gold was also observed, indicating that a high degree of localization was possible for the effects.

Unlike photosensitizers, the nanoparticles and microparticles used for these studies can remain stable and inert in cells for extended periods, and hence may be useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner.

Reference:

Pitsillides, C. M.; Joe, E. K.; Wei, X.; Anderson, R. R.; and Lin C. P.: Selective cell targeting with light-absorbing microparticles and nanoparticles. Biophys. J., 84, 4023-4032 (2003).

Abstract (courtesy of the Biophysical Journal):
http://www.biophysj.org/cgi/content/abstract/84/6/4023

Gold cluster labels have been investigated for a number of applications in cancer research and therapy, including the use of radioactive gold conjugates targeted to cancer. Hainfeld has reviewed this field:

Hainfeld, J. F.: Gold, electron microscopy, and cancer therapy. Scanning Microsc., 9, 239-254; discussion 254-256 (1995).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8553020&dopt=Abstract

More information:

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The Further Adventures of Nanogold® in Neuroscience

Hereditary spastic paraplegias (HSPs) comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset HSP SPG3A is caused by mutations in the atlastin/hGBP3 gene (atlastin1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein (GBP) superfamily of large GTPases. Immunofluorescence showed that atlastin1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus.

Nanogold® labeling with HQ Silver enhancement proved highly effective in localizing this protein at the subcellular level. Primary cultures of rat cortical neurons were prepared from E18 rat embryos. After 6 days in culture, they were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min, washed with 0.1 M phosphate buffer, permeabilized and blocked in 5% normal goat serum (NGS) with 0.1% saponin in phosphate-buffered saline (PBS) for 1 h, then incubated with anti-atlastin1 antibodies in blocking buffer for 1 h. After washing with 1% NGS-PBS and 2% non-fat milk in PBS, cells were incubated with Nanogold anti-rabbit secondary antibody (1:250) in 2% non-fat milk/PBS for 1 h. After washing with 2% non-fat milk/PBS, the cells were then fixed with 2% glutaraldehyde in PBS for 30 min, thoroughly washed with PBS and distilled water, and silver enhanced with HQ Silver. After washing again in water and 0.1 M phosphate buffer, the cells were treated with 0.2% osmium tetroxide in 0.1 M phosphate buffer for 30 min, en bloc mordanted with 0.25% uranyl acetate in acetate buffer (pH 5.0) overnight, washed and dehydrated in serial concentrations of ethanol, and finally infiltrated and embedded in epoxy resins. Thin sections of ~70 nm were counter-stained with uranyl acetate and lead citrate.

A predominant localization of atlastin1 to the cis-Golgi was found; further studies showed that atlastin1 can self-associate, and most likely exists as a tetramer in vivo. Membrane fractionation and protease protection assays revealed that atlastin1 is an integral membrane protein with two predicted transmembrane domains. These findings suggest that the SPG3A protein atlastin1 is a multimeric, integral membrane GTPase involved in Golgi membrane dynamics or vesicle trafficking, and is required for correct axon growth: it is possible that disruption by atlastin1 gene mutations may result in impaired axon growth during neuronal development, leading to HSP SPG3A.

Reference:

Zhu, P. P.; Patterson, A.; Lavoie, B.; Stadler, J.; Shoeb, M.; Patel, R., and Blackstone, C.: Cellular Localization, Oligomerization, and Membrane Association of the Hereditary Spastic Paraplegia 3A (SPG3A) Protein Atlastin. J. Biol. Chem., Sep 23 [Epub ahead of print: M306702200v1] (2003).

More information:

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Other Recent Publications

Want to do in situ hybridization (or other labeling procedures) on plant nuclei? Lavania and group describe an enzymatic protocol for the isolation of nuclei from root tip tissue that avoids the damage caused by mechanical processes to remove cell wall materials. The protocol utilizes selective harvest of active nuclei from root tip tissue in liquid suspension under the influence of cell wall-degrading enzymes: 3% (v/v) pectinase from Aspergillus niger, and 2% (w/v) cellulase (i.e., 1.8% cellulase from Aspergillus niger and 0.2% Onozuka RS cellulase), incubated at 37°C for 2050 min. Cell wall free nuclei at a given stage of cell cycle, free of any cell debris, could be realized in suspension that are fit for preparation of extended fibers suitable for fiber FISH applications.

Reference:

Lavania, U. C.; Yamamoto, M., and Mukai, Y.: Extended Chromatin and DNA Fibers from Active Plant Nuclei for High-resolution FISH. J. Histochem. Cytochem., 51, 1249-1253 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/10/1249

A series of articles focuses on the principles and recent developments at the intersection of nanotechnology and biotechnology, or 'nanobiotechnology,' this week. Among them, Zhang reviews the fabrication of biomaterials by molecular self-assembly, illustrated by his work on the preparation of nanofibers and nanotubules from peptides, proceeding to the construction of 3-dimensional arrays from 10 nm nanotubes.

Reference:

Zhang, S.: Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol., 21, 1171-1178 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14520402&dopt=Abstract

Mirkin and co-workers continue their nanotechnology communications with a method for the growth of silver nanoparticles of controlled size and shape. Using a narrow-band light source, in this case a 150W xenon lamp fitted with a 550 nm, 40 nm width bandpass filter, they produced triangular silver nanoprisms from a suspension of 10 nm colloidal silver particles; the method was found to produce relatively narrow size distributions with edge lengths in the 30 - 120 nm range.

Reference:

Jin, R.; Cao, Y. C.; Hao, E.; Metraux, G. S.; Schatz, G. C.; and Mirkin, C. A.: Controlling anisotropic nanoparticle growth through plasmon excitation. Nature, 425, 487-490 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14523440&dopt=Abstract

With Nam and Thaxton, Mirkin has also described a biological 'bar-code' assay in which magnetic nanoparticles are used to tag a target protein with its genetic marker, which is then separated, amplified and detected. The process, as demonstrated using prostate-specific antigen (PSA) produces an increase in sensitivity of six orders of magnitude over that required for clinical diagnosis, detecting 30 attomolar concentrations of PSA. The assay comprises magnetic microparticles conjugated with antibodies against the target of interest, and nanoparticles functionalized with both antibodies that can bridge to the bound target, and DNA unique to the target. Complexed particles are isolated magnetically, then the DNA dehybridized and detected either using 30 nm silver-enhanced gold nanoparticles on chips, or by PCR followed by ethidium bromide staining in gel electrophoresis or scanometric detection.

Reference:

Nam, J. M.; Thaxton, C. S.; and Mirkin, C. A.: Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science, 301, 1884-1886 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14512622&dopt=Abstract

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