Updated: September 10, 2003
N A N O P R O B E S E - N E W S
Vol. 4, No. 9 September 10, 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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We are very pleased to introduce a new reagent, Nickel (II) nitriloacetic acid (NTA) Nanogold®, a new general-purpose labeling and staining reagent which targets polyhistidine (His) tags. Ni-NTA-Nanogold may be used to localize expressed His-tagged targets by electron microscopy, light microscopy and in blotting applications.
Features of Ni-NTA-Nanogold include:
- The nitrilotriacetic acid - Ni(II) chelate is much smaller than an antibody, producing a probe with unprecedented resolution, ideal for localizing sites in proteins and protein complexes at molecular resolution. The small size also means that it is better able to access restricted sites and penetrate into specimens.
- Ni-NTA-Nanogold is made with a modified gold particle, stabilized in a slightly different manner to the Nanogold used for other labeling reagents and conjugates, which results in very high solubility and stability. This 1.8 nm particle is also readily visualized by electron microscopy.
- Although small, binding constants for Ni(II)-NTA are high; the chelate effect of multiple histidine binding and multiple Ni(II)-NTA functionalization gives dissociation constants estimated from 10-7 to 10-13 M-1. This provides binding strengths comparable to antibodies for many applications.
References:
- Hainfeld, J. F.; Liu, W.; Halsey, C. M. R.; Freimuth, P., and Powell, R. D.: Ni-NTA-Gold Clusters Target His-Tagged Proteins. J. Struct. Biol., 127, 185-198 (1999).
- Abstract (courtesy of the Journal of Structural Biology)
- Buchel, C.; Morris, E.; Orlova, E., and Barber, J.: Localisation of the PsbH subunit in photosystem II: a new approach using labelling of His-tags with a Ni(2+)-NTA gold cluster and single particle analysis. J. Mol. Biol., 312, 371-379 (2001).
- Abstract (courtesy of the Journal of Molecular Biology)
More information:
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A paper describing preliminary results using a combined Nanogold® and Texas Red-labeled dextran as a neuronal tracer, presented at Microscopy & Microanalysis '03 in San Antonio, Texas last month, is a new addition this month to the 'Research Applications' section of our web site. This novel reagent was prepared by the conjugation of Mono-Sulfo-NHS-Nanogold to a 10,000 MW amino-functionalized dextran already conjugated with Texas Red.
As described by Dr. Eduardo Rosa-Molinar of the University of Puerto Rico-Rio Piedras, it was then used to retrogradely label spinal motor neurons innervating a median unpaired fin, the sexually dimorphic anal fin musculature in female and male Western Mosquitofish, Gambusia affinis affinis. The sexually dimorphic anterior transposition of the median unpaired fin, specifically the anal fin, provides a versatile experimental model for studying intercellular mechanisms, processes, and interactions during post-embryonic development, especially changes of the nervous system as the animal changes from a non-internal fertilizing to an internal fertilizing species. Retrograde tract tracing using this and other probes revealed a unique spinal cord region associated with the 12th through 14th vertebrae, a portion of the unique ano-urogenital region, containing a population of secondary motor neurons with extensive dendritic arborization; these segments also contain a plexus innervating the muscles of the median unpaired fins. The female G. a. affinis was shown to have fewer and smaller secondary motor neurons than did males, and the neurons branching and dendritic arborization were more reduced than those in males.
This application has been discussed before using polyamindoamine dendrimers, or branched-chain polymers, to carry the fluorescent and gold labels, and this work is briefly described in our paper presented at Microscopy & Microanalysis '98. Dextrans possess the advantages of higher solubility and are also relatively inexpensive.
More information:
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Do our silver enhancement reagents work with gold probes from other suppliers? Absolutely! HQ Silver works well with any colloidal gold conjugate. It is the only commercially available silver enhancement reagent to contain a protective colloid: this enables it to produce highly uniform particle sizes and growth rates, while its low ionic strength and near-neutral pH results in high levels of ultrastructural morphology preservation.
Shirahama-Noda and group demonstrate this in their recent paper, in which they use HQ Silver to enhance colloidal gold. As part of their studies on the physiological
role of asparaginyl endopeptidase (AEP)/legumain (an asparagine-specific cysteine proteinase) in mammals, they used anti-AEP primary antibodies and 10 nm gold-conjugated secondary antibodies to localize localization AEP in ultrathin cryosections of mouse kidney. They found that AEP was localized to lamp-2-positive late endosomes, which were merged with greatly enlarged lamp-2-positive membrane structures. This, combined with the observation of completely defective processing of cathepsins B, H, and L from the single-chain forms into the two-chain forms in the deficient mice, confirmed the pivotal role of AEP in the endosomal/lysosomal degradation system.
Mice were deeply anesthetized and fixed by vascular perfusion with 2.5% glutaraldehyde, 0.2% picric acid, and 0.1 M sodium phosphate (pH 7.4) at a room temperature. The removed kidneys were fixed a further 2h in the same fixative, then incubated overnight in 0.1 M sodium phosphate buffer, pH 7.4, containing 2.3 M sucrose and 20% polyvinyl pyrrolidone. 6 micron-thick sections were cut on a cryostat, picked up on Formvar-carbon-coated nickel grids, incubated with 2% gelatin in PBS containing 10 mM glycine, then reacted with anti-AEP antibodies followed by colloidal gold-conjugated secondary antibodies. The gold labeling was intensified with HQ Silver. The cryosections were then dehydrated, and embedded in LR White resin.
References:
- Shirahama-Noda, K.; Yamamoto, A.; Sugihara, K.; Hashimoto, N.; Asano, M.; Nishimura, M, and Hara-Nishimura, I.: Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice. J. Biol. Chem., 278, 33194-33199 (2003).
- Abstract (courtesy of the Journal of Biological Chemistry)
- Koh, S.; Yamamoto, A.; Inoue, A.; Inoue, Y.; Akagawa, K.; Kawamura, Y.; Kawamoto, K., and Tashiro, Y.: Immunoelectron microscopic localization of the HPC-1 antigen in rat cerebellum. J. Neurocytol., 22, 995-1005 (1993).
More information:
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Constructs of oligonucleotides linked to gold nanoparticles are the subject if intense current interest due to their applications in nanobiotechnology. Recent papers provide new insights into the mechanisms by which these species interact, and considerations for the design of oligonucleotide-gold nanoparticle constructs.
Sandstrm and group studies the interactions of thiol-modified and non-thiolated oligonucleotides with 13 nm colloidal gold particles, and found binding in both instances, suggesting other binding mechanisms in addition to thiol coordination. Previous studies have attributed this to charge-based interactions between the exposed bases and the colloidal gold particles, which possess an overall negative charge; however, Sandström reports that non-specific binding is significant even with double-stranded DNA, as shown by binding isotherms and circular dichroism, and instead proposes that it arises through ion-induced dipole dispersive interactions, where the negatively charged phosphate groups on the DNA induce dipoles in the highly polarizable gold particles. Mobility experiments supported different binding modes for the two types on interactions: the thiol-modified oligonucleotides projected more from the surface, resulting in increased friction and reduced mobility, while unmodified oligonucleotides lay flat on the surface, giving reduced friction and higher mobility. Particles with nonspecifically adsorbed DNA could be separated from the specifically modified and unmodified ones by gel electrophoresis.
Reference:
Sandström, P.; Boncheva, M., and Åkerman, B.: Nonspecific and Thiol-Specific Binding of DNA to Gold Nanoparticles. Langmuir, 19, 7537-7543 (2003).
Article information (courtesy of the American Chemical Society):
http://dx.doi.org/10.1021/la034348u
Alivisatos reports that non-specific binding is absent in complexes of thiol-modified DNA with 5 nm gold particles, perhaps due to the reduced polarizability of the smaller gold particles.
Reference:
Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D., and Alivisatos, A. P.: Electrophoretic Isolation of Discrete Au Nanocrystal/DNA Conjugates. Nano Lett., 1, 32-35 (2001).
Article information (courtesy of the American Chemical Society):
http://dx.doi.org/10.1021/nl005508e
Kumar and co-workers have investigated the formation of complexes between alkylamines and gold nanoparticles. Results are presented on the Fourier transform infrared spectroscopy, thermogravimetry, nuclear magnetic resonance, and X-ray photoemission (XPS) characterization of 5 nm gold nanoparticles capped with the alkylamines laurylamine and octadecylamine, prepared by phase transfer of aqueous gold nanoparticles to chloroform containing fatty amine molecules. Thermogravimetry and XPS analysis of purified powders of the amine-capped gold nanoparticles indicated the presence of two different modes of binding of the alkylamines with the gold surface. The weakly bound component is attributed to the formation of an electrostatic complex between protonated amine molecules and surface-bound [AuCl4]-/[AuCl2]- ions, while the more strongly bound species is tentatively assigned to a complex of the form [AuCl(NH2R)].
Reference:
Kumar, A.; Mandal, S.; Selvakannan, P. R.; Pasricha, R.; Mandale, A. B., and Sastry, M.: Investigation into the Interaction between Surface-Bound Alkylamines and Gold Nanoparticles. Langmuir, 19, 6277-6282 (2003).
Article information (courtesy of the American Chemical Society):
http://dx.doi.org/10.1021/la034209c
Meanwhile, Ge and co-workers report the assembly of DNA on dodecanethiol-coated 11 nm colloidal gold particles prepared by phase transfer; in this case, the mechanism of interaction was thought to be hydrophobic interactions.
Reference:
Ge, C.; Liao, L.; Wang, Y.; Chen, K., and Gu, N.: DNA Assembly on 2-Dimensional Array of Colloidal Gold. Biomedical Microdevices, 5, 157-162 (2003).
Abstract (courtesy of Kluwer Academic Publishers):
http://ipsapp007.kluweronline.com/content/getfile/4554/16/8/abstract.htm
These results suggest that any of these types of interactions may disrupt selective covalent conjugation reactions. Achieving selective conjugation without interference from these non-specific interactions requires the use of gold particles stabilized with a non-ionizing, hydrophilic shell. Our 1.4 nm Nanogold particles satisfy these requirements, and have been used for a number of selective conjugation reactions to produce oligonucleotide conjugates with interesting and potentially useful properties. We are currently developing larger gold particles with similar properties: a prototype is described in our 1999 Microscopy & Microanalysis paper.
More information:
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Looking for a complete price list in a handy format? Look up prices and catalog numbers for all our products at a glance with our current price list, available on our web site as a PDF file. Alternatively, if you'd prefer to order through a distributor, look up your national or regional distributor on our international distributor page.
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Kamnev and co-workers describe the application of surface-enhanced infrared spectroscopy - the spectroscopic observation of the enhancement of specific vibrational modes upon conjugation to colloidal metal particles - as a method for immunochemical detection. Using surface-enhanced infrared absorption (SEIRA) spectroscopy of dry films of colloidal gold (CG) bioconjugates with protein A, certain characteristic bands of the protein (e.g., amide I, amide II and some other vibration modes) are essentially affected by the metal surface; it was also found that the reaction of protein A attached to colloidal gold particles with human immunoglobulin G (IgG) results in further essential changes in SEIRA spectra, providing a means for an easy and rapid IR spectroscopic detection.
Reference:
Kamnev, A. A.; Dykman, L. A.; Tarantilis, P. A., and Polissiou, M. G.: Spectroimmunochemistry Using Colloidal Gold Bioconjugates. Biosci. Reports, 22, 541-547 (2002).
Abstract (courtesy of Kluwer Academic Publishers):
http://ipsapp007.kluweronline.com/content/getfile/4551/27/7/abstract.htm
Khlebtsov and group have presented a theoretical study of an optical model of conjugates of colloidal gold nanoparticles (5 - 200 nm) with biopolymers, analyzed in terms of two-layer spherical particles with a gold core and dielectric coating. The Mie theory was used to study the dependence of variations (caused by the adsorption of a biopolymer on the particle surface) in the extinction and light scattering (at 90°) spectra on the gold core diameter, shell refractive index and thickness.
Reference:
Khlebtsov, N. G.; Dykman, L. A.; Bogatyrev, V. A., and Khlebtsov, B. N.: Two-Layer Model of Colloidal Gold Bioconjugates and Its Application to the Optimization of Nanosensors. Colloid J., 65, 508517 (2003).
Abstract (courtesy of Kluwer Academic Publishers):
http://ipsapp007.kluweronline.com/content/getfile/4605/17/18/abstract.htm
Nolting and co-workers report the preparation of a 2 nm gold glycoconjugate from disulfides containing C-glycosides with galactosyl and glucosyl headgroups linked to triethylene glycol via an amide bond, as part of continuing studies of the molecular events involved in HIV recognition of mucosal membrane cells - in this case, the interactions of the HIV-associated glycoprotein gp120, with cellular receptor GalCer.
Reference:
Nolting, B.; Yu, J.-J.; Liu, G.; Cho, S.-J.; Kauzlarich, S., and Gervay-Hague, J.: Synthesis of Gold Glyconanoparticles and Biological Evaluation of Recombinant Gp120 Interactions. Langmuir, 19, 6465-6473 (2003).
Article information (courtesy of the American Chemical Society):
http://dx.doi.org/10.1021/la034414s
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