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Updated: November 19, 2002

N A N O P R O B E S     E - N E W S

Vol. 3, No. 11          November 18, 2002


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 [email protected].

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New Products: Combined Alexa Fluor®* and Nanogold® Probes

We are pleased to announce that Alexa Fluor® 488 and 594 FluoroNanogold probes are now available. Benefit from the improved performance and additional features of the super-bright Alexa Fluor probes:
  1. Increased fluorescence signal and higher quantum yield - ideal for scarce targets or dynamic systems where exposure needs to be restricted.

  2. Fluorescence remains high and consistent across wider pH range.

  3. Improved solubility means reduced non-specific interactions, lower background and higher signal-to-noise ratios.

  4. Uses fluorescein (Alexa Fluor® 488) or Texas Red (Alexa Fluor® 594) filter sets.

  5. Available in 1 mL or affordable 0.5 mL sizes.

In addition, with Alexa Fluor® 594, you can differentiate a FluoroNanogold-labeled target from a second target labeled with fluorescein, Alexa Fluor® 488, green fluorescent protein, or other fluorophores.

More information:

*Alexa Fluor is a registered trademark of Molecular Probes, Inc.

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Fluorescence Properties: Combined Fluorescent and Gold Conjugates

What happens to the fluorescence of fluorophores when a gold particle is nearby? A number of theories have been proposed, which informed the design and development of our combined fluorescent and gold probes. In our 1998 paper, we used the Frster theory to show that fluorescence was largely preserved in combined fluorescent and gold nanoparticle conjugates, provided that the separation of the fluorescent and the gold labels is greater than the Frster distance (about 5 nm with Nanogold®):

Powell, R. D.; Halsey, C. M. R., and Hainfeld, J. F.: Combined fluorescent and gold immunoprobes: Reagents and methods for correlative light and electron microscopy. Microsc. Res. Tech., 42, 2-12 (1998).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9712158&dopt=Abstract

At shorter separation distances, other mechanisms may ensure even greater quenching, and this is useful for other appllications, in particular molecular beacons. Dubertret and co-workers have found that Nanogold®, used as a quencher in molecular beacons, is significantly more effective than the conventional DABCYL quencher, yielding signal-to-noise ratios (for open : closed configuration) of up to 1,000 or higher:

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

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

Nanoprobes is currently developing new technology for this application. Dulkeith and co-workers now report their investigations of the effect on radiative and non-radiative fluorescence lifetimes for systems in which fluorophores were linked to metal nanoparticles of varying sizes from 1 to 30 nm. They found both an increase in the radiative lifetime, and a decrease in the non-radiative lifetime, both of which contribute to quenching; this implies that fluorescence quenching by attached gold particles is greater than predicted by Frster theory alone. With a gold-fluorophore separation of 1 nm, they find about 99.8 % quenching even with 1 nm particles:

Dulkeith, E.; Morteani, A. C.; Niedereichholz, T.; Klar, T. A.; Feldmann, J.; Levi, S. A.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Enschede, A. E.; Mller, M., and Gittins, D. I.: Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects. Phys. Rev. Lett., 89, 203002 (2002).

Abstract (courtesy of Physical Review Letters):
http://ojps.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=PRLTAO000089000020203002000001&idtype=cvips&gifs=Yes

More information - prototype combined fluorescent and metal nanoparticle probes:

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Conductimetric Sensors using Gold Nanoparticles and Metal Deposition

Metal nanoparticles are one of the building blocks of nanotechnology, and also potential templates for the fabrication of nanoscale components by autometallography. Mirkin and co-workers have reported the fabrication of a conductimetric biosensor based on both processes. The device comprises lithographically prepared microelectrode pairs (separation 20 micrometers), a shorter capture oligonucleotide strand located in the gap between them, and a longer target oligonucleotide in solution. The target oligonucleotide has contiguous recognition elements that are complementary to the capture strand on one end and on the other to oligonucleotides attached to Au nanoparticles: when the device with the pair of electrodes is immersed in a solution containing the appropriate probe and target, gold nanoparticle probes fill the gap, and when these are silver enhanced, the gap becomes conductive. Reference:

Park, S.-J.; Taton, T. A., and Mirkin, C. A.: Array-Based Electrical Detection of DNA with Nanoparticle Probes. Science, 295, 1503 (2002).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11859188&dopt=Abstract

The site- and chemically-specific reactivity of Nanogold® give a new level of synthetic control over the preparation of oligonucleotides, enabling labeling at specific thiol or amine sites, yielding greater control over chemical nanofabrication; alternatively, the affinity of positively charged Nanogold for negatively charged macromolecules may be used as an organizing mechanism for the formation of molecular wires. In one example, DNA strands were used as templates to deposit positively charged Nanogold particles, which may then be linked to form conductive wires by autometallography. In addition, our unique gold enhancement process, which deposits gold instead of silver, may be used to improve conductivity.

More information:

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What is the Weight of Nanogold®? Can I Use it on Gels?

Nanogold® is a gold cluster complex containing between 55 and 75 gold atoms, coated with a mixture of tris (aryl) phosphine and halide ligands which coordinate to surface gold atoms. Its molecular weight is close to 15,000. However, because very dense gold atoms comprise about two-thirds of this, the molecule is actually smaller than a protein or other macromolecule of the same weight. In size-selective processes, such as gel filtration, Nanogold frequently elutes similarly to a protein of about 8,000. Therefore, its separation from larger macromolecules may be greater, and its separation from smaller ones less, than expected.

We are frequently asked whether Nanogold-labeled molecules may be separated using gel electrophoresis. While there is evidence that this may be useful, we advise caution in interpreting the results, as gel shifts alone are frequently not found to be good indicators of labeling. Although Nanogold® has a nominal MW of 15,000, in practice the observed gel shift may be very small.

If you wish to use this method, we recommend the following:

  1. Use non-reducing gels: thiol-containing reagentsd such as dithiothreitol (DTT) can break down the Nanogold cluster.

  2. Detect each component separately and specifically to confirm labeling. Separate the reaction mixture or product, divide the gel, then stain one portion with silver enhancement (note: do not use silver protein stain, but a silver enhancement reagent specific for gold particles, such as LI Silver from Nanoprobes) or gold enhancement, and the other with a general protein stain such as Coomassie blue. You may be able to resolve two bands, one which develops with silver enhancement *and* Coomassie (the Nanogold conjugate) and one of which develops only with Coomassie (unlabeled protein). The presence of unbound Nanogold would add a third band which reacts only with silver.

  3. When silver or gold enhancing to detect the Nanogold conjugate, monitor the progress of development carefully. This is a highly sensitive detection method, and in addition to the target band, a "tail" may also develop behind it which, if left too long, may obstruct resolution of the band.

For some illustrations, see p. 92-94 of Hainfeld and Furuya:

Hainfeld, J. F., and Furuya, F. R.: Silver Enhancement of Nanogold and Undecagold. In "Immunogold-Silver Staining: Principles, Methods and Applications," M. A. Hayat (Ed.); CRC Press, Boca Raton, FL, 1995, pp. 71-96.

An application note, "Detection of Nanogold®-labeled molecules on Gels," is available on our web site:

www.nanoprobes.com/App1A.html

We find that gel filtration is the most effective method for isolating most Nanogold conjugates. For detailed help in designing separation procedures and in selecting appropriate media, see our Guide to Gold Cluster Labeling:

More information:

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Other Recent Publications

Harbord and co-workers give a helpful protocol for using Nanogold conjugates in post-embedding labeling for electron microscopy of peritoneal neutrophils. Wild-type neutrophils were fixed in 2% paraformaldehyde containing 0.5% glutaraldehyde in PBS, dehydrated through a graded series of alcohol, followed by two changes of propylene oxide, and embedded with araldite CY212 resin.

Reference:

Harbord, M.; Novelli, M.; Canas, B.; Power, D.; Davis, C.; Godovac-Zimmermann, J.; Roes, J., and Segal, A. W.; Ym1 Is a Neutrophil Granule Protein That Crystallizes in p47 phox -deficient Mice. J. Biol. Chem., 277, 5468-75 (2002).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/277/7/5468

Kambe and co-sorkers, meanwhile, have used non-fluorescent Nanogold® in conjunction with Immunofluorescence staining for fluorescence and electron microscopic immunohistochemistry; double immunofluorescent labeling for ZnT-5 and insulin was conducted using paraffin sections from paraformaldehyde-fixed human pancreas; for electron microscopy, the paraformaldehyde-fixed cells were rinsed in phosphate-buffered saline and processed by the pre-embedding silver-intensified immunogold method using Nanogold anti-mouse IgG followed by silver enhancement (HQ silver); specimens were then osmificated, dehydrated, and embedded in Quetol 812 and ultrathin sections stained with an aqueous solution of 2% uranyl acetate for observation.

Reference:

Kambe, T.; Narita, H.; Yamaguchi-Iwai, Y.; Hirose, J.; Amano, T.; Sugiura, N.; Sasaki, R.; Mori, K.; Iwanaga, T., and Nagao, M.: Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells. J. Biol. Chem., 277, 19049-55 (2002).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/277/21/19049

Sompuram and co-workers describe a novel quality control technology that may may be valuable in creating standardized controls to quantify IHC analytical variability.Standardized antibody targets are created and attached to the same slides as the patient sample; after IHC staining, the targets turn the same color as the stained cells or tissue elements. Unlike current clinical practice, these targets are neither cells nor tissue sections, but short constrained peptides attached directly to the glass slide, and simulate the portion of the native antigen to which the antibody binds.

Reference:

Sompuram, S. R.; Kodela, V.; Zhang, K.; Ramanathan, H.; Radcliffe, G.; Falb, P., and Bogen, S. A. A Novel Quality Control Slide for Quantitative Immunohistochemistry Testing. J. Histochem. Cytochem., 50, 1425-1434 (2002).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/10/1381

Using Nanogold®, antibodies or antibody fragments for the detection of haptenated nucleic acid targets may be labeled site-selectively for greater retention of activity; Nanogold may also be conjugated directly to chemically derivatized oligonucleotides.

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