Updated: October 7, 2004

N A N O P R O B E S     E - N E W S

Vol. 5, No. 10          October 7, 2004

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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Nanogold® Enhances Radiation Therapy of Cancer

Gold cluster labels and nanoparticles can be used in a number of ways for cell- and tissue- specific therapeutics. For example, we recently reported on the use of targeted gold nanoparticles used with laser illumination to inactivate cells in a highly selective manner. Radioactive gold conjugates have been targeted to cancer using antibodies.

At Nanoprobes, we have now extended gold-based cancer therapy to the in vivo application of gold nanoparticles. Can they be used for diagnosis and therapy? For example, gold absorbs X-rays more strongly than most tissue, and causes more dose to be locally deposited. If a tumor could be specifically loaded with gold, the tumor dose would be increased, and radiotherapy would be enhanced.

Mice bearing EMT-6 mammary carcinomas received a single intravenous injection of 1.9 nm-diameter gold particles (Nanogold-X), up to 2.7 g Au/kg body weight, which elevated concentrations of gold to 7 mg [Au]/g in tumors. Tumor-to-normal-tissue gold concentration ratios remained ~8:1 during several minutes of 250 kVp X-ray therapy. One-year survival was 86% versus 20% with X-rays alone and 0% with gold alone. The increase in tumors safely ablated was dependent on the amount of gold injected. The gold nanoparticles were apparently non-toxic to mice and were largely cleared from the body through the kidneys. This novel use of small gold nanoparticles permitted achievement of the high metal content in tumors necessary for significant high-Z radioenhancement.

References:

More information:

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Nanogold®: Charge and Thermal Sensitivity

Some issues raised during recent technical questions deserve a wider audience:

What is the isoelectric point of Nanogold®?

Unlike colloidal gold, Nanogold® is not stabilized by proteins or other macromolecules. Apart from the specific chemical groups introduced either to give reactivity (maleimido-, Sulfo-N-hydroxysuccinimide) or to impart specific properties (positive or negative charge), the gold surface is coated with small organic molecules (ligands) whose outer ends are capped by p-N-methylcarboxamidophenyl groups. These provide solubility in aqueous buffers and in many other solvents, and because they are not protonated or deprotonated by pH changes, do not introduce charge. Overall, the structure is very similar to that of undecagold (right), except that the gold core is larger.  

[Undecagold] (3k)]

Structure of Monomaleimido Undecagold, showing coordinated ligands and maleimide group.

Apart from the amines or carboxyls specified for each product, Nanogold does not contain any functionalities that contribute to ionization. None of our Nanogold products contain both positively and negatively ionizing groups: therefore, they do not exhibit isoelectric behavior, and do not have an isoelectric point.

Monoamino Nanogold and Positively Charged Nanogold contain respectively close to one, and several (estimated 3-6) primary aliphatic amines. These therefore are expected to have very similar ionizing properties to compounds such as n-propylamine, or ethyl 3-aminopropyl-carboxamide. Thus, it would be positively ionized at low pH values, but increasingly neutral at higher pH values, and close to neutral above about pH 9. Negatively Charged Nanogold contains multiple (about 6-12) carboxylic acid groups; these are in the form of p-carboxylatophenyl groups, and therefore the compound will have very similar ionizing properties to benzoic acid. Thus, it will be negatively charged at higher pH values, but increasingly neutral at lower pH values, and close to neutral below about pH 3. Mono-Sulfo-NHS-Nanogold contains a sulfo- group, and is therefore permanently negatively ionized in aqueous solution.

One exception to this is NTA-Ni(II)-Nanogold. The NTA ligand contains both a tertiary aliphatic amine and three carboxyl groups, and therefore will exhibit isoelectric behavior in the absence of complexed coordinated nickel (II) ion. However, while this ion is present, both the amine and carboxyls are coordinated to it.

You instructions contain a caution on the use of elevated temperatures. How sensitive is Nanogold and its conjugates to high temperatures? How does this affect embedding procedures using resins that are cured at high temperatures, and experiments that may require elevated temperatures?

Although we do caution users about elevated temperatures, we have shown that Nanogold under normal conditions is generally quite resistant to high temperatures. For example, Nanogold in aqueous solution has been heated to close to 100°C for several hours and demonstrates minimal decomposition, as monitored by UV/visible spectroscopy (see Hainfeld and Furuya, reference below).

Thermal stability is usually only a problem when another complicating factor is present. We have found that the following conditions can render Nanogold more sensitive to elevated temperatures:

  • Low pH (below about 3-4).
  • High ionic strengths (higher than about 0.3 M).
  • Presence of a thiolated reducing agent such as dithiothreitol (DTT), mercaptoethylamine (MEA) or mercaptoethanol.

If none of these factors are present, the Nanogold probe should tolerate elevated temperatures well, and will usually work well in embedding procedures at 37°C, 50°C or 60°C. However, we do recommend that for periods longer than a few hours conjugates are kept at room temperature or below, and for longer than a few days they should be refrigerated.

Reference:

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.

More information:

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Localizing Nuclear Scaffold Components with Nanogold®

The small size and high sample penetration of Nanogold® conjugates, and their consequent ability to access and label nuclear and other hard-to-reach antigens, was demonstrated recently by Kireeva, Belmont and group, who used Nanogold labeling to determine the distribution of scaffold components in mitotic chromosomes and develop a model for mitotic chromosome structure. The distribution of two scaffold components, topoisomerase II and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), were correlated with the appearance of prophase chromosome folding intermediates using fluorescence microscopy; ultrastructural localization was then determined by pre-embedding immunogold staining.

Log phase HeLa cells were treated with 600 ng/ml nocodazole (2 hours). Mitotic cells collected by three shake-offs, separated by 15 minutes, put on ice between shake-offs, then washed in PBS, and incubated in 75 mM KCl for 10 minutes. Cells were centrifuged, resuspended in buffer A (80 mM KCl, 20 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 15 mM PIPES, 0.5 mM spermidine, 0.2 mM spermine, and 10 microgram/mL turkey egg white inhibitor, pH 7.0) with 0.1 mg/ml digitonin, and vortexed 2 x 30 s to release chromosomes. Cells and nuclei were pelleted by centrifugation, and the supernatant applied to glass coverslips. Chromosomes were adhered by low speed centrifugation, then placed into buffer C (0.25 M sucrose, 10 mM Pipes, 1.5 mM MgCl2, 1.0 mM CaCl2 and 10 micrograms/mL turkey egg white protease inhibitor, pH 6.8) and fixed in 1.85% freshly prepared paraformaldehyde for 10 min. Coverslips were washed in buffer C (3 x 5 minutes), buffer A (3 x 5 minutes), buffer A with 20 mM glycine (3 x 5 minutes), then blocked in 6% normal goat serum in buffer A (1 hour). Staining was conducted using rabbit anti-hCAP-E/SMC2 peptide primary antibody used at 0.23 micrograms/mL, or mouse monoclonal anti-topoisomerase II-alpha primary antibody (Topogen) at 1:500 dilution in buffer A, followed by Nanogold goat antirabbit secondary antibody (1:400 dilution) for 20 hours at 4°C. Coverslips were washed in buffer C and fixed in 2% glutaraldehyde for 2 h. Silver enhancement was done as described by Burry and Gilerovitch, followed by embedding in Epon.

Axial distributions of both topoisomerase II and SMC2 were confirmed in unextracted metaphase chromosomes, with SMC2 localizing to a 150 to 200 nm diameter central core, but contrast to the predictions of current radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses, which then resolve into linear, 200-300 nm diameter middle prophase chromatids that double in diameter by late prophase. These results support a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."

Reference:

Kireeva, N.; Lakonishok, M.; Kireev, I.; Hirano, T., and Belmont, A. S.: Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure. J. Cell Biol., 166, 775-785 (2004).

Silver enhancement procedures:

  • Gilerovitch, H. G.; Bishop, G. A.; King, J. S., and Burry, R. W.: The use of electron microscopic immunocytochemistry with silver-enhanced 1.4nm gold particles to localize GAD in the cerebellar nuclei. J. Histochem. Cytochem., 43, 337-343 (1995).

  • Burry, R. W.: Pre-embedding immunocytochemistry with silver-enhanced small gold particles. In Immunogold-Silver Staining: Principles, Methods, and Applications.; M. A. Hayat (Ed.), CRC Press, Boca Raton, FL. pp. 217230 (1995).

More information:

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Gold-Conjugated DNAzymes for Colorimetric Lead Detection

The color change from red to blue that occurs when gold particles are aggregated in solution has formed the basis for a number of methods for detecting biological targets. Mirkin reported this color change when gold particles functionalized with complementary oligonucleotides were aggregated by hybridization. More recently, Li and Rothberg described the use of 13 nm gold particles for the rapid colorimetric detection of PCR products: mixing the gold particles with an annealed PCR product mixture resulted in the adsorption of the annealed strands to the gold particles, and re-hybridization on cooling resulted in aggregation and a color change from red to blue-gray.

Liu and Lu now report the application of this method to the development of a biosensor for the colorimetric detection of metal ions, specifically Pb2+, in solution. Their system exploits the recently discovered catalytic properties of some DNA sequences, known as deoxyribozymes or DNAzymes, which can selectively cleave target sequences in the presence of metal ions. The recognition element in the biosensor is the Pb2+-specific DNAzyme known as the "8-17" DNAzyme, which comprises a substrate strand (17DS) and an enzyme strand (17E). Both strands are DNA, except that the substrate strand contains a single RNA linkage (ribonucleoside adenosine, (rA)) that serves as the cleavage site. This is extended at each end by a different 12-nucleotide DNA sequence: signaling is provided by 42 nm gold nanoparticles functionalized with 3'- or 5'- thiol-modified oligonucleotides complementary to the ends of the probe. Annealing and hybridization gives a blue color; however, in the presence of Pb2+, the linking ribonucleotide is cleaved, the particles no longer aggregate, and the blue color no longer develops.

By optimization of the component concentrations, reaction conditions, and their selection of a sensor design and nanoparticle size that allowed observation of hybridization at room temperature, the requirement for heating and cooling was eliminated, resulting in a biosensor that could produce a result in as little as 10 minutes. A linear response was observed for Pb2+ concentrations from 0.4 to 2 x 10-6 M, and interference from other metal ions was not observed.

Reference:

Liu, J., and Lu, Y.: Accelerated Color Change of Gold Nanoparticles Assembled by DNAzymes for Simple and Fast Colorimetric Pb(2+) Detection. J. Amer. Chem. Soc., 126, 12298-12305 (2004).

The covalent conjugation chemistry of Nanogold® provides many options for conjugation to oligonucleotides, giving potential advantages for the construction of oligonucleotide-based molecular structures. More information:

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Nanoprobes ISH Technology at the Brisbane IAP Meeting

If you are attending the XXV Congress of the International Academy of Pathology in Brisbane, Australia on October 10-15, find out more about recent developments in our detection technologies, including enzyme metallography, and their application to in situ hybridization detection in a presentation by Dr. Raymond R. Tubbs, our collaborator at the Cleveland Clinic Foundation, in the special evening session sponsored by Ventana Medical Systems (Note: the conference web site doesn't work in Netscape).

More information:

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Other Recent Publications

The range of quantum dots available was recently extended by the addition of lead selenide nanoparticles. With a band gap of 0.26 eV at room temperature, PbSe nanoparticles have the capacity to produce photoluminescence in the mid-infrared. Hollingsworth, Pietryga and co-workers now report the synthesis of colloidal PbSe nanoparticles that possess with efficient, particle-size-tunable, narrow bandwidth mid-IR photoluminescence (PL) at energies as low as 0.30 eV. 8 nm and larger lead selenide nanoparticles were prepared by reaction of lead(II) acetate trihydrate and oleic acid in trioctylphosphine (TOP) and phenyl ether by heating under vacuum, followed by reaction with TOP selenium in phenyl ether (10 mL) at 200°C - 250°C: nanoparticles were isolated by rapid cooling. PL intensity was significantly increased by passivation with cadmium selenide, suggesting that these new quantum dots may be functionalized similarly to other semiconductor nanoparticles.

Reference:

Pietryga, J. M.; Schaller, R. D.; Werder, D.; Stewart, M. H.; Klimov, V. I., and Hollingsworth, J. A.: Pushing the Band Gap Envelope: Mid-Infrared Emitting Colloidal PbSe Quantum Dots. J. Amer. Chem. Soc., 126, 11752-11753 (2004).

Meanwhile, Chen, Zheng and co-workers have described a novel photosensitizing molecular beacon, which is based upon peptide recognition rather than oligonucleotide hybridization. The beacon consists of a cleavable peptide caspase 3 substrate, linked at one end to pyropheophorbide (a chlorophyll analogue that has long-wavelength absorption at 667 nm and is an efficient singlet oxygen producer with a quantum yield > 50%) as the photosensitizer, and at the other to a carotenoid (CAR), an efficient scavenger and quencher of singlet oxygen, in a configuration holding these two entities in close proximity. Enzyme binding and cleavage was then detected by luminescence and lifetime measurements of the singlet oxygen produced once the CAR was cleaved.

Reference:

Chen, J.; Stefflova, K.; Niedre, M. J.; Wilson, B. C.; Chance, B.; Glickson, J. D., and Zheng, G.: Protease-triggered photosensitizing beacon based on singlet oxygen quenching and activation. J. Amer. Chem. Soc., 126, 11450-11451 (2004).

Are you planning correlative microscopy of bone? If so, a recent report by Laboux et al. may be helpful for sample preparation and processing. They have found that microwave irradiation improves the preservation of bone by ethanol. Samples were dehydrated for 7 hours in 70% ethanol with three 5-minute cycles of microwave irradiation, then embedded in methylmethacrylate (MMA) at 4°C. Immunohistochemical localization of osteopontin using the Dako Envision enzyme polymer detection system, and post-embedding immunoelectron microscopy labeling of osteopontin using a chicken egg yolk-anti-OPN primary antibody and colloidal gold-labeled anti-chicken secondary antibody, could be performed on the same sample. Morphological preservation was found to be improved in those specimens subjected to the microwave irradiation, compared with controls dehydrated for one week in 70 % ethanol.

Reference:

Laboux, O.; Dion, N.; Arana-Chavez, V.; Ste-Marie, L. G., and Nanci, A.: Microwave Irradiation of Ethanol-fixed Bone Improves Preservation, Reduces Processing Time, and Allows Both Light and Electron Microscopy on the Same Sample. J. Histochem. Cytochem., 52, 1267-1275 (2004).

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