Updated: January 11, 2002

N A N O P R O B E S E - N E W S

Vol. 3, No. 1 January 11, 2002

This monthly newsletter is 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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Control of Dna Hybridization through Inductive Coupling to Nanogold®

Hamad-Schifferli and co-workers at the Massachusetts Institute of Technology Media Lab have demonstrated that the highly localized temperature rise produced by a conjugated Nanogold® particle inductively couple to a pulsed radio frequency magnetic field may be used for the remote control of DNA hybridization. Mono-Sulfo-NHS Nanogold was used to label molecular beacons with a primary amino- group synthetically inserted into the loop. The researchers also developed a system to determine the extent of the local temperature rise: a surface-bound Nanogold-oligonucleotide antenna was hybridized to a fluorescently labeled complementary strand. Correlation of the concentration of dissociated fluorescent probe with temperature with and without application of the magnetic field indicated a local temperature rise of 13°C; furthermore, the effect was highly localized, and neighboring molecules lacking the Nanogold antenna were unaffected. The effect is fully reversible. Reference:

Hamad-Schifferli, K.; Schwartz, J. J.; Santos, A. T.; Zhang, S., and Jacobson, J. M.: Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna. Nature, 2002, 415, 152-155.

Abstract (requires registration):
http://www.nature.com/nlink/v415/n6868/abs/415152a_fs.html

Nanogold has already been shown to be a highly effective quencher for molecular beacons (hairpin loops of DNA with a fluorescent group at one end and a quencher at the other: when they bind to their target, they open, the fluorophore and the quencher move apart, and fluorescent signal appears). Nanogold® has been found to improve the "signal-to-noise ratio" (the ratio of fluorescence intensity when the beacon is open to when it is closed) from 100 to up to several thousand. Reference:

Dubertret, B., Calame, M., and Libchaber, A.: Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol., 2001, 19, 365-370.

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11283596&dopt=Abstract

Product and technical information about the conjugation of Nanogold to oligonucleotides:

Technical help with DNA labeling: http://www.nanoprobes.com/TechNGlr.html#DNA
Catalog information - Nanogold labeling reagents: http://www.nanoprobes.com/LabRgts.html
Other References: http://www.nanoprobes.com/RefTopNG.html#Ndna

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GoldEnhance Enhancement of Immunolabeling on Metal Substrates

Silver-enhanced immunogold labeling of specimens on metal substrates is problematic. Although smaller gold particles produce much more successful labeling, the metal substrates promote severe etching of silver enhancement by osmium. Owen and co-workers now report that GoldEnhance, a new Autometallography reagent from Nanoprobes that deposits gold instead of silver, is not affected. They successfully labeled vinculin in the focal adhesion sites of fibroblasts using 5 nm gold with gold enhancement, and observed labeling by SEM. Reference:

Owen, G. R.; Meredith, D. O.; Ap Gwynn, I., and Richards, R., G.: Enhancement of immunogold-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions. Cell. Biol. Int., 2001, 25, 1251-1259.

Abstract (Ideal):
http://www.idealibrary.com/links/doi/10.1006/cbir.2001.0846
Other publications by the authors:
http://www.aofoundation.org/ari/research/interface/publications.shtml

More information about gold enhancement:

Introduction and catalog information: http://www.nanoprobes.com/GoldEnhance.html
Our 1999 MSA paper: http://www.nanoprobes.com/MSAGE99.html
Technical help: http://www.nanoprobes.com/TechGE.html
Other references: http://www.nanoprobes.com/Refge.html

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Improved Immunolabeling with Nanogold®-Labeled Single-Chain Variable Fragment (Scfv) Antibodies

Thought Nanogold-Fab' was the smallest gold immunoprobe you could get? You can use Nanogold® labeling reagents to prepare even smaller gold-labeled antibody fragments. Malecki and co-workers have described the preparation of recombinant single-chain variable fragment (scFv) antibodies labeled with Monomaleimido Nanogold via a cysteine residue. Nanogold-labeled anti-biotin Fab' and scFv fragments were used as secondary probes against a biotinylated primary against Pex3 to label peroxisomes in yeast cells: the scFv resulted in significantly higher labeling density than the Fab' conjugate. Reference:

Malecki*, M.; Hsu, A.; Truong, L., and Sanchez, S: Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains; Proc. Natl. Acad. Sci. USA, 2002, 99, 213-218.

Abstract (PNAS):
http://www.pnas.org/cgi/content/abstract/99/1/213

Ribrioux and co-workers have also used fluorescent and Nanogold-labeled Fv fragments to demonstrate enhanced penetration and labeling. Reference:

Ribrioux, S., Kleymann, G., Haase, W., Heitmann, K., Ostermeier, C., and Michel, H.: Use of Nanogold- and Fluorescent-labeled Antibody Fv Fragments in Immunocytochemistry. J. Histochem. Cytochem., 44, 207-213 (1996).

Abstract (Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/44/3/207

Protocols for Monomaleimido Nanogold: http://www.nanoprobes.com/Inf2020.html
Protocols for Mono-Sulfo-NHS Nanogold: http://www.nanoprobes.com/Inf2025.html
References for Nanogold labeling by application: http://www.nanoprobes.com/RefTopNG.html
Guide to Gold Cluster Labeling: http://www.nanoprobes.com/LGuide.html

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DNA Nanowires and Gold Cluster Crystals

A number of other approaches may be taken to putting gold onto DNA, and for using the organizing properties of DNA to form larger arrays of gold particles. We have recently reproduced our 2001 paper from Microscopy & Microanalysis 2001, in which we used DNA strands as templates to deposit positively charged Nanogold® particles, on our web site. These may then be linked to form molecular wires by the autometallographic deposition of silver (silver enhancement) or gold (gold enhancement).

Our paper: http://www.nanoprobes.com/MSADNA01.html
Charged Nanogold: http://www.nanoprobes.com/LabRgts.html#charged
Gold enhancement: http://www.nanoprobes.com/GoldEnhance.html

In a second paper, from Microscopy & Microanalysis 2000, we describe the formation and Scanning Transmission Electron Microscope observation of proto-crystalline arrays of a gold cluster compound, dubbed "Greengold," which is similar in size to Nanogold:

Our paper: http://www.nanoprobes.com/MSAXTALS00.html

To make navigation easier, we have recently divided the "Applications" section on our web site into two sections: "Product Applications," which describe applications of our current products, and "Research Applications," which describe new technologies we are developing. Visit our applications index for a closer look:

http://www.nanoprobes.com/Applic.html

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Other Recent Publications

A new silver-gold intensified in situ hybridization method for electron microscopy is described by Liem and co-workers, who used it for the ultrastructural localization of intramuscular expression of BDNF mRNA. They amplified the DAB reaction product from peroxidase labeling with silver followed by gold for EM observation. Reference:

Liem, R. S. B. ; Brouwer, N., and Copray, J. C. V. M.: Ultrastructural localisation of intramuscular expression of BDNF mRNA by silver-gold intensified non-radioactive in situ hybridisation. Histochem. Cell Biol., 2001, 116, 545-551.

Abstract (Springer):
http://link.springer.de/link/service/journals/00418/contents/01/00349/

Desnoyers and co-workers report a novel method for the fluorescent localization of receptors in tissue sections, using a recombinant insulin-like growth factor (IGF-I) chimera incorporating a six-histidine tag, followed by a biotinylated antibody, then fluorescein-labeled streptavidin. This was compared with immunolabeling of IGF-I using a secondary Texas Red-labeled antibody. Reference:

Desnoyers, L.; Simonette, R. A.; Vandlen, R. L., and Fendly, B. M.: Novel Non-isotopic Method for the Localization of Receptors in Tissue Sections. J. Histochem. Cytochem., 2001, 49, 1509-1518.

Abstract (Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/49/12/1509

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