N A N O P R O B E S     E - N E W S

Vol. 6, No. 2          February 11, 2005

One area of concern is distinguishing genuine amplification of a gene from polysomy, in which chromosome duplication results in more copies of the gene than usual. Differentiating the two requires counting the individual copies, and in order to be useful for clinical applications, a detection method must be both sufficiently sensitive to detect each gene copy, and sufficiently precise that it may be distinguished from its neighbors. It is also important that other stains may be used, allowing the detection of a second target either to confirm the result, or as a control.

Recently, we have developed a new detection method, enzyme metallography, with several advantages for in situ hybridization:

• High sensitivity combined with very low background, giving a clean signal.
• High resolution and minimal diffusion give sharp, highly resolved signals from individual gene copies.
• Signals are opaque and black, and hence readily distinguished from other stains.
• Brightfield adaptation uses more accessible conventional light microscope, with no dark adaptation required.
• Underlying tissue morphology is observed simultaneously for more complete interpretation.

Schematic of silver or gold-enhanced Nanogold-based in situ hybridization detection (left) and enzyme metallography (right).

Having optimized the method for in situ hybridization, Ray Tubbs and our collaborators at the Cleveland Clinic have incorporated both gene and protein detection into a single assay directed to assessing malignancy of breast cancer. By using in situ hybridization with enzyme metallography detection for the HER2 gene, and immunohistochemistry with fast red K for the corresponding HER2 oncoprotein, they have developed a two-color assay for HER2 status that simultaneously shows gene and protein status. The method has excellent correlation with either method run alone, and clearly differentiates the different degrees of gene amplification.

Reference:

Tubbs, R.; Pettay, J.; Hicks, D.; Skacel, M.; Powell, R.; Grogan, T., and Hainfeld, J.: Novel bright field molecular morphology methods for detection of HER2 gene amplification. J. Mol. Histol., 35, 589594 (2004).

• Abstract (courtesy of the SpringerLink).

Enzyme metallography has many more potential applications: in light microscopy, it may be used for immunohistochemistry and immunocytochemistry, and it also produces highly sensitive results in immunoblots and even in the detection of targets on lateral flow strips. The deposited metal is also readily visualized by electron microscopy, making it a potential detection and visualization method for correlative microscopy.

More information:

• Enzyme Metallography for correlative labeling (our Microscopy & Microanalysis 2004 abstract)
• Enzyme Metallography - a New staining method (our Microscopy & Microanalysis 2002 abstract)
• Product application special report: Nanogold in situ hybridization
• Other product and research applications

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Immuno-EM with Tyramide Signal Amplification and Nanogold® Detection

In the latest issue of the Journal of Histochemistry and Cytochemistry, Lee and co-workers describe the use of this method for the electron microscopy immunostaining of GM130, a cis-Golgi matrix protein, in a cell line: it was found to be highly sensitive and more enhanced than that a comparable protocol lacking the amplification step.

A human melanoma cell line, G361, was cultured in RPMI supplemented with 10 % fetal bovine serum (FBS) and fixed in 3.0 % glutaraldehyde in 0.1 M phosphate buffer. The fixed cells were immersed in 1 % sodium borohydride in phosphate-buffered saline (PBS) to block free aldehydes. Cells were quenched in 3 % H₂O₂ in 60 % methanol to inactivate endogenous peroxidase, then incubated in blocking buffer (10 % normal horse serum, 1 % bovine serum albumin and 0.1% gelatin in PBS). In the simple (non-amplified) protocol, the cells were incubated in anti-GM130 antibody (1.0 micrograms/mL or 0.1 micrograms/mL), followed by biotinylated secondary antibody (1:200 dilution) then StreptavidinNanogold (1:100) in incubation buffer (10 % normal horse serum, 1 % bovine serum albumin and 0.1 % gelatin in PBS). For the TSA protocol, the signal was amplified using the following sequence of reagents: primary antibody (0.1 micrograms/mL; anti-GM130 Ab), biotinylated secondary antibody (1:200), streptavidinhorseradish peroxidase (HRP) (diluted 1:500 in 10 % normal horse serum, 1 % BSA and 0.1 % gelatin in TBS (0.15 M NaCl, 50 mM Tris-HCl, pH 7.5)), biotinyltyramide (diluted 1:50 in 1 x amplification diluent), and StreptavidinNanogold (1:100). Cells were then enhanced using HQ silver and gold toning, followed by Epon infiltration and examination in the electron microscope.

The simple protocol resulted in specific gold particle labeling over the cis-side of Golgi apparatus; omission of the primary antibody led to very low background labeling. Preservation of the cellular ultrastructure was fairly good and many organelles, including nucleus, mitochondria, and Golgi apparatus, could be recognized. The TSA protocol was then carried out using a more diluted primary antibody (10 times less than the recommended use). Both the TSA technique and the non-amplified protocol yielded a particulate black signal over the Golgi apparatus. However, the signal somewhat more intense with the TSA technique. Gold particle distribution was quite limited within the Golgi apparatus in both cases: the signal comprised a collection of gold particles along the ribbons of the Golgi cisternae, while the nucleus and mitochondria were devoid of labeling. One interesting observation was that the particles themselves were larger with the TSA method, suggesting perhaps the fusion of multiple closely-spaced particles during the enhancement process.

Reference:

Lee, S.-W.; Lee, S. E.; Ko, S. H.; Hong, E. K.; Nam, K.-I, Nakamura, K.; Imayama, S.; Park, Y.-J.; Ahn, K. Y.; Bae, C. S.; Kim, B. Y., and Park S. S.: Introduction of Tyramide Signal Amplification (TSA) to Pre-embedding Nanogold-Silver Staining at the Electron Microscopic Level. J. Histochem. Cytochem., 53, 249-252 (2005).

• Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

Kohler, A.; Lauritzen, B., and Van Noorden, C. J.: Signal amplification in immunohistochemistry at the light microscopic level using biotinylated tyramide and nanogold-silver staining. J. Histochem. Cytochem., 48, 933-941 (2000).

• Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).
• Reprint (PDF) (courtesy of the Journal of Histochemistry and Cytochemistry).

More information:

• Catalog information: Nanogold conjugates
• Product information: Nanogold-Streptavidin
• Protocol for Nanogold detection with tyramide signal amplification
• Technical Help: Nanogold conjugates
• Technical Help: Immunoelectron microscopy

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Dividing Nanogold® for Multiple Reactions

Monomaleimido Nanogold and Mono-Sulfo-NHS-Nanogold are both supplied lyophilized from 0.02 M sodium phosphate buffer with 150 mM sodium chloride, at a pH appropriate to their reactivity (6.5 for Monomaleimido Nanogold, 7.5 for Mono-Sulfo-NHS-Nanogold). This is because the reactive groups are hydrolyzed in a few hours in aqueous solution, and therefore must be supplied dry to preserve these groups.

Once reconstituted in aqueous solution, these reagents will only be active for about 1-2 hours. If you need to conduct multiple reactions with one vial, there are two options:

• Divide the reagents based on weight; however, this is not ideal since the quantities are very small, and the lyophilized compounds can form thin films that are difficult to divide and remove. However, if you wish to try this approach, the amounts of solid (including the buffer salts) present in each vial are as follows:

  Labeling reagent	Catalog number and size	Weight of material	Catalog number and size	Weight of material
  Monomaleimido Nanogold	2020 (30 nmol size)	11.65 mg	2020A or 2020S (6 nmol size)	2.33 mg
  Mono-Sulfo-NHS-Nanogold Nanogold	2025 (30 nmol size)	11.94 mg	2025A or 2025S (6 nmol size)	2.39 mg
  Monoamino Nanogold	2021 (30 nmol size)	0.45 mg	2021A or 2021S (6 nmol size)	0.09 mg
  Charged Nanogold	2022 and 2023 (30 nmol size)	0.45 mg	N/A	N/A

• Dissolve the reagents in a small amount of anhydrous aprotic solvent and partition by volume. DMSO (dimethylsulfoxide) is an excellent solvent for Nanogold and is well tolerated by most biological molecules. Once separated, the aliquots may then be refrigerated for as long as several days, then immediately before use diluted to the volume required with deionized water. We do not recommend DMF (N,N-dimethylformamide) as a solvent since it has occasionally formed a white precipitate with Nanogold; however, DMA (N,N-dimethylacetamide) has very similar properties and is an excellent solvent for Nanogold.

I accidentally left the reagents at the wrong temperature. Will they be harmed?

The critical factor with our Nanogold labeling reagents is exposure to water, not temperature. Provided the vials have not been opened (and hence exposed to water), the reagents will be fine if they have been left at room temperature for several days or even a couple of weeks.

For our Silver enhancement and gold enhancement reagents, the principal aging mechanism is through reaction with oxygen. Provided the vials have not been opened for a prolonged time (and hence exposed to atmospheric oxygen), storage at room temperature for a few days (up to a week) will not harm them. Likewise, freezing LI Silver or GoldEnhance will not harm the reagents. However, a small amount of the materials may precipitate. If you do accidentally freeze these reagents, allow them to warm to room temperature for a few hours, then agitate gently to redissolve any precipitate.

More information:

• Technical Help: Nanogold labeling reagents
• Technical Help: Silver and Gold Enhancement
• Guide to Gold Cluster Labeling
• Guide: Spectra and labeling calculation
• Catalog Information: Nanogold labeling reagents
• Catalog Information: Silver enhancers
• Catalog Information: Gold enhancement

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More on the Preparation and Activity of Gold Nanoparticles

While this work is very interesting, we would be grateful if the authors could get our name right; we are Nanoprobes, Incorporated, not "The Nanogold Company."

Reference:

Lee, K.-M.; Park, S.-T., and Lee, D.-J.: Nanogold synthesis by inert gas condensation for immuno-chemistry probes. J. Alloys Compounds, 390, 297300 (2005).

Abstract (courtesy of Science Direct).

Meanwhile, Willner, Zayats and co-workers (featured in last month's newsletter) now report the growth of gold nanoparticles catalyzed by hydrogen peroxide, and its application to the development of a glucose sensor based on the biocatalytic enlargement of gold nanoparticles. Addition of H₂O₂ to a 0.01 M phosphate buffer solution containing [AuCl₄]^- (2 x 10^-4 M) to citrate-stabilized gold nanoparticle seeds (12 ± 1 nm: 3 x 10^-10 M), with 2 x 10^-3 M cetyltrimethylammonium (CTAC) as surfactant, resulted in an immediate increase of the electronic absorbance corresponding to the gold nanoparticle plasmon. As the concentration of H₂O₂ increases, the absorbance is intensified. Control experiments showed that no Au NPs are formed in the absence seeds, and that added H₂O₂ is required for the absorbance changes. These results suggest that the gold nanoparticle seeds act as catalysts for the reduction of [AuCl₄]^- by H₂O₂, resulting in enlargement of the particles and enhanced spectral absorbance features.

A closer inspection of the spectral changes showed a red shift (ca. 15 nm) at low H₂O₂ concentrations, and a blue shift at higher H₂O₂ concentrations; while the red shift may correspond to enlargement of the particles, the blue shift may correspond to the formation of either more smaller nanoparticles, or a mixture of very small crystalline nanoparticles together with enlarged ones. HR-TEM analysis of the enlargement process revealed that the gold nanoparticles exist in several morphologies (spheres, rhombs, triangles, and polygons) with a very narrow size distribution, 12 ± 1 nm. Particles formed upon treatment of the seeds with low (5 x 10^-5 M) and a high (2.4 x 10^-4 M) H₂O₂ concentration in the presence of [AuCl₄]^-/CTAC (for 5 min), respectively, were found to contain nanoparticles with a strong dark contrast of dimensions corresponding to ca. 13 nm x 13 nm, coated by numerous nanocrystallites of lighter contrast, that yield Au NP clusters of dimensions up to ca. 18 nm x 27 nm. At the higher H₂O₂ concentration, the nanocrystallite-coated nanoparticles reached larger dimensions, up to ca. 32 nm x 28 nm, but in addition, numerous small separated gold nanocrystal flakes of 2.5 to 7 nm were also observed. The majority of the observed flakes contained dislocations, and part of them were found to be folded and to appear as two-dimensional crystallites. The Au crystallites are catalytically grown at the intersection of the faces of the parent seed Au NPs. At low H₂O₂ concentrations the flakes are mainly associated with the parent nanoparticles, leading to structurally enlarged particles and red-shifted absorbance; at high H₂O₂ concentrations, a mixture of small Au nanocrystallites together with enlarged nanoparticles is generated: the increased nanoparticle content yields the high absorbance, but the mixture of different sized particles leads to the observed blue shift of the absorbance spectra.

Many oxidase enzymes generate H₂O₂ upon the oxidation of their substrates by molecular oxygen. This suggests that H₂O₂-mediated catalytic growth of gold nanoparticles may be used for the optical detection of their substrates. To test this hypothesis, the group investigated the change in gold nanoparticle spectra in a model system using glucose oxidase, GOx, and O₂/glucose as the H₂O₂ generating system with 3 x 10^-10 M gold nanoparticle seed solution, including [AuCl₄]^-/CTAC. Upon the addition of different concentrations of glucose, the absorbance values of the particles increased as the concentration of glucose was raised; control experiments indicate that no growth occurs upon exclusion of glucose, GOx, or O₂. These results indicate that the biocatalyzed formation of H₂O₂ is essential for the growth of the particles, and confirms that this provides a method for detecting enzymatic action.

Reference:

Zayats, M.; Baron, R.; Popov, I., and Willner, I.: Biocatalytic Growth of Au Nanoparticles: From Mechanistic Aspects to Biosensors Design. Nano Lett., 5, 21-25 (2005).

• Abstract (courtesy of the American Chemical Society).
• Reprint (courtesy of the American Chemical Society).

More information:

• Nanoprobes: Research and technology applications
• A covalent 10 nm gold immunoprobe (our Microscopy & Microanalysis 1999 abstract)
• Catalog Information: Nanogold labeling reagents
• Gold-based autometallography (our Microscopy & Microanalysis 1999 abstract)
• References sorted by topic: Nanogold labeling

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Nanoprobes at USCAP

• Yoder, B. J.; Choueiri, T.; Downs-Kelly, E.; Skacel, M.; Hainfeld, J.; Powell, R.; Roche, P.; Grogan, T.; Pettay, J.; Budd, T.; Hicks, D. G., and Tubbs, R. R.: EnzMet GenePro, a Novel Bright Field Assay that Simultaneously Profiles HER-2 Genotype and Phenotype, Correlates with Overall and Disease Free Survival of Breast Cancer. Poster session II (Breast Pathology), #247: Monday, February 28, 2005, 1:00 - 4:30 PM; Convention Center, Exhibit Hall B.

• Tubbs, R. R.; Pettay, J.; Roche, P. C.; Powell, W.; Powell, R. D.; Grogan, T., and Hainfeld, J. F.: Enzyme Metallography (EnzMet) - A Robust Detection System for High Resolution Ultrasensitive Immunohistochemistry (IHC). Poster session III (Techniques), #1556: Tuesday, March 1, 2005, 9:30 AM - 12:00 Noon; Convention Center, Exhibit Hall B.

More information:

• USCAP Meeting: Full details
• USCAP Meeting: Abstract search (requires registration)
• Catalog Information: Nanogold conjugates
• Nanogold labeling: References, sorted by application

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Other Recent Publications

Reference:

Danscher, G., and Stoltenberg, M.: Zinc-specific Autometallographic In Vivo Selenium Methods: Tracing of Zinc-enriched (ZEN) Terminals, ZEN Pathways, and Pools of Zinc Ions in a Multitude of Other ZEN Cells. J. Histochem. Cytochem., 53141-153 (2005).

• Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

Danscher, G.: Exogenous selenium in the brain. A histochemical technique for light and electron microscopical localization of catalytic selenium bonds. Histochemistry, 76, 281293 (1982).

• Abstract (courtesy of PubMed (Medline)).

Platinum nanoparticles are the target for Huang and co-workers, who in this month's Langmuir describe how to prepare them using the hydrosilylation reaction. The hydrosilylation reaction, an addition of a hydrosilane unit (Si-H) to a double bond (C=C) to form an alkylsilane, is widely utilized in silicon polymers engineering; it may be initiated in several ways, one of the most common being the use of platinum-based catalysts is the Karstedt catalyst (platinum divinyltetramethyldisiloxane complex). During the platinum-catalyzed hydrosilylation reaction, colloidal platinum is formed; while previously regarded as a nuisance, this is actually an effective preparation method for stabilized platinum nanoparticles. In a typical synthesis, the concentration of platinum divinyltetramethyl-disiloxane complex in the reaction solution was 1 mM. Predetermined amounts of hydrosilane and olefin were placed in a 50-mL Schlenk flask with a magnetic stirrer, and the flask charged by anhydrous toluene (10 mL) and evacuated and refilled with nitrogen three times. Platinum divinyltetramethyldisiloxane complex (2 mM solution, 10 mL) was added by a syringe, and the reaction stirred under nitrogen at 60°C. The reaction solution became colored, then turned dark brown after 72 hours, indicating the formation of platinum nanoparticles. Depending on the ligands used, the particles could be produced in sizes from 1.9 to 4.5 nm.

Reference:

Huang, J.; Liu, Z.; Liu, X.; He, C.; Chow, S. Y., and Pan, J.: Platinum Nanoparticles from the Hydrosilylation Reaction: Capping Agents, Physical Characterizations, and Electrochemical Properties. Langmuir, 21, 699-704 (2005).

• Article information (courtesy of the American Chemical Society).

If surface-enhanced Raman spectroscopy (SERS) is your thing, Lu and co-workers report a novel advance. SERS is the measurement of vibrational spectra in which certain modes are intensified by adsorption of the molecules to silver nanoparticles. Lu and colleagues have developed a method for preparing tunable substrates by control of the interparticle spacing. The high-density nanoparticle thin film is prepared by self-assembly of 20 nm silver nanoparticles, with oleylamine and oleic acid as the capping reagents, via the Langmuir-Blodgett (LB) technique on a water surface and transferring the particle monolayer to a shrinkable temperature-responsive polymer membrane, in this case Poly (N-isopropylacrylamide) (PNIPAM). This allows the preparation of a dynamic surface enhanced Raman scattering substrate: the plasmon peak of the silver nanoparticle film red shifts up to 110 nm with increasing temperature, and the scattering signal enhancement factor can be dynamically tuned by the thermally activated SERS substrate.

Reference:

Lu, Y.; Liu, G. L., and Lee, L. P.: High-Density Silver Nanoparticle Film with Temperature-Controllable Interparticle Spacing for a Tunable Surface Enhanced Raman Scattering Substrate. Nano Lett., 55-9 (2005).

• Abstract (courtesy of the American Chemical Society).
• Reprint (courtesy of the American Chemical Society).

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