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Updated: February 11, 2005

N A N O P R O B E S     E - N E W S

Vol. 6, No. 2          February 11, 2005

In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

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Enzyme Metallography for In Situ Hybridization

Our page on Nanogold® in situ hybridization has been among the most popular pages on our web site for a while now, and the Nanogold-based detection method, which can detect single gene copies, is among the most sensitive available. However, we have continued to develop new and better technology for in situ hybridization detection that addresses current clinical issues.

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.

[ISH Detection methods: schematic] (42k)]

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.


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).

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:

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Immuno-EM with Tyramide Signal Amplification and Nanogold® Detection

While tyramide signal amplification (TSA) was one of the enabling factors in the use of Nanogold® for ultrasensitive in situ hybridization, it is equally applicable to the detection of scarce antigens in immunostaining procedures, at both the light and electron microscopy level: and in both cases, detection of the amplification product using Nanogold with autometallography can be used for a unique combination of ultrasensitive detection with very high resolution.

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.


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).

TSA with silver-enhanced Nanogold detection for light microscopy:

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).

More information:

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Dividing Nanogold® for Multiple Reactions

More from our list of the questions we are most often asked

I have to do multiple reactions, but don't need five aliquots, I need even less than 6 nmol, or I need different quantities for several reactions. How can I divide the Nanogold® labeling reagents?

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:

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More on the Preparation and Activity of Gold Nanoparticles

Several papers have recently investigated alternative ways to make gold particles, how to conjugate them, and the properties of molecules that are linked to them. Lee, Park and Lee describe the preparation of approximately 1 nm particles, very similar to Nanogold®, by a materials engineering process, inert gas condensation (IGC): metals are evaporated inside an ultra-high vacuum chamber filled with helium or argon gas at a low pressure, typically a few hundred pascals. The evaporated metal atoms lose their kinetic energy through interatomic collisions with the gas atoms, and condense in the form of small crystals, which accumulate, because of convective flow, on a vertical liquid nitrogen-filled cold finger.

gold wire (about 0.5 g) was evaporated on a heat resistant tungsten boat in an argon atmosphere after evacuating the chamber to 1.0610-3 Pa with a diffusion pump. The processing input current was changed from 50 to 65A. Evaporation temperature was measured using a K-type thermocouple, and chamber pressure controlled by a mass flow controller (MFC) using argon gas with a range of 7266 Pa. Evaporation time was within a 2050 s range depending on the input current. Gold nanoparticles were directly collected on a copper grid used as a TEM sample holder exposed for 13 s near a cold finger, then examined by field-emission transmission electron microscopy. At a temperature of 1,124°C and pressure of 133 Pa, nanoparticles approximately 1 nm in size were formed; at different temperatures (both higher and lower) or lower pressures, larger particles were formed.

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."


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.


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).

More information:

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Nanoprobes at USCAP

We will be presenting new results from our project to develop new chromogenic detection and staining methods for HER2 gene amplification detection and immunohistochemical staining at the upcoming USCAP Meeting in San Antonio. Look for the following presentations:

  • 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:

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Other Recent Publications

Gorm Danscher and colleagues review the selenium-based autometallographic tracing of zinc in this month's Journal of Histochemistry and Cytochemistry. The selenium method was introduced in 1982 as a tool for zinc-ion tracing. If sodium selenide/selenite is injected into the brain, spinal cord, spinal nerves containing sympathetic axons, or intraperitoneally, retrograde axonal transport of zincselenium nanocrystals takes place in ZEN neurons. As a result, accumulation of zincselenium nanocrystals occurs in lysosomes of the neuronal somata; this is therefore also a highly specific tool for tracing ZEN pathways. This review updates the original paper and presents evidence that if the protocols are followed strictly, only zinc ions are traced.


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).

Original paper:

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

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.


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).

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.


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).

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