N A N O P R O B E S     E - N E W S

Vol. 8, No. 4          April 30, 2007

Updated: April 30, 2007

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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GoldEnhance and Nanogold® Double Labeling

Gold enhancement is an alternative to silver enhancement, developed by Nanoprobes. With gold enhancement, gold nanoparticles, colloidal gold or gold cluster labels are enlarged by the catalytic deposition of gold, rather than silver, from solution.

Gold enhancement provides the same degree of sensitivity as silver enhancement, but it has several important advantages:

  • Gold enhancement may safely be used before any strength osmium tetroxide - gold enhanced particles are not etched as silver can be.
  • May be used in physiological buffers without the risk of precipitation (including chlorides, which precipitate silver).
  • Unlike silver enhancement, gold enhancement may be conducted in the presence of metals (such as metallic substrates for cell culture or biomaterials).
  • The metallographic reaction is less pH sensitive than that of silver.
  • Gold gives a much stronger backscatter signal than silver, making it better for SEM labeling applications.
  • GoldEnhance is near neutral pH for best ultrastructural preservation.
  • Low viscosity, so the components may be dispensed and mixed easily and accurately.

GoldEnhance: how it works [(37k)]

Enhancement of Nanogold by GoldEnhance: mechanism. Final particle size is controlled by enhancement time.

In their recent paper in Cell, Wang, Paspalas and co-workers describe the use of Nanogold® with gold enhancement for the localization of biological components that are important for spatial working memory (WM, or "scratchpad" memory). WM is maintained by spatially tuned, recurrent excitation within networks of prefrontal cortical (PFC) neurons, evident during delay periods in WM tasks. The authors used a double labeling method where two Nanogold labeling reactions were used with sequential gold enhancement steps, one after labeling of the first target with Nanogold and the second after labeling the second target, to give two populations of gold particles with non-overlapping sizes. In this way two components of the PFC neuron arrays, postsynaptic a2A adrenoceptors (alpha-2A-AR) and Cyclic Nucleotide-gated channels (HCN1/HCN2) were localized and their spatial relationship established.

Monkey PFC tissue was processed for dual immunolabeling using combinations of enzymatic and/or gold-based immunotechniques, and reversal of the immunocytochemical sequence. Primary antibodies in tris-buffered saline with 2% normal goat serum (N-TBS) were simultaneously applied for 48 hours at 4°C. To visualize the first target, sections were preincubated for 30 minutes in N-TBS supplemented with 0.1% acetylated BSA (Aurion), 0.1% fish skin gelatin and 0.07% Tween 20 (i.e., gold buffer), then transferred for 3 hours to Nanogold-conjugated goat anti-rabbit Fab' (1:200 in gold buffer). After washing in ultrapure water and 20 mM sodium citrate, the Nanogold was enhanced for 12 minutes on ice with using GoldEnhance EM. Subsequently, Nanogold-conjugated goat anti-mouse Fab' (1:200) was applied for 4 hours to probe the second target bound with primary antibody. Sections were finally postfixed in glutaraldehyde and transferred for 4 minutes to the gold developer to enhance the second target, and also to enhance further (i.e., 12 + 4 minutes in total) the gold signal of the first series (two-step autometallography). This sequential enhancement produced distinct, nonoverlapping particle-size groups.

An alternative gold and peroxidase double labeling method was also used. In this procedure, the second target was labeled using species-specific bridging antibodies and peroxidaseantiperoxidase tertiary complexes (1:200 in N-TBS for 2 hr each). Peroxidase activity was visualized in 0.025% diaminobenzidine (DAB) in TBS with the addition of 0.007% hydrogen peroxide. In this case, gold enhancement for the first label ranged from 3 to 8 min at 22° C. Sections were processed for electron microscopy and layers IIII of area 46 were sampled for analysis in the transmission electron microscope at 80 kV.

This study demonstrates that HCN1 or HCN1/HCN2 heteromers on spines of pyramidal dendrites are spatially coexpressed with the alpha-2A-AR, thus providing a potent substratum for functional interaction in the primate PFC. Electrophysiological and cognitive experiments support a model where alpha-2A-AR agonists such as guanfacine improve PFC cognitive function by inhibiting the production of cAMP, closing HCN channels, and strengthening the PFC networks that underlie delay-related cell firing in monkeys performing a spatial WM task. These data reveal a powerful mechanism for rapidly altering the strength of WM networks in PFC.


  • Wang, M.; Ramos, B. P.; Paspalas, C. D.; Shu, Y.; Simen, A.; Duque, A.; Vijayraghavan, S.; Brennan, A.; Dudley, A.; Nou, E.; Mazer, J. A.; McCormick, D. A., and Arnsten, A. F.: alpha-2A-Adrenoceptors Strengthen Working Memory Networks by Inhibiting cAMP-HCN Channel Signaling in Prefrontal Cortex. Cell, 129, 397-410 (2007).

More information:

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Nanogold® Labeling Reagents: Avoiding Aggregation

Aggregation can be a problem when labeling peptides and smaller proteins with Nanogold®. While there are several possible reasons for this, the most likely culprit is the interaction of the Nanogold particle with hydrophobic domains on the protein or peptide, particularly if the protein has hydrophobic domains. Another possibility is cross-linking through thiols; this may be the case if your peptide or protein contains more than one thiol, particularly if it is prepared by the reduction of a disulfide in the native molecule and is easily re-oxidized.

If you are observing aggregation and believe it arises from solubility issues, the following solutions may be helpful:

  • Use an ispropanol-water mixtures (20% isopropanol) for labeling, provided that your peptide or protein can tolerate this; this will tend to solubilize hydrophobic domains better than aqueous buffers along. Although it generates heat when mixed with water, DMSO is another useful organic solvent, as Nanogold is highly soluble. If you plan to use DMSO, dissolve your protein in aqueous solution containing 20% DMSO (provided, again, that your protein can tolerate it), then add the reconstituted Nanogold.

  • Addition of a small amount of the detergent Tween-20 to the Nanogold reaction buffer also helps to prevent hydrophobic interactions. In some cases a non-ionic detergent such as CHAPS may be advisable in order to better preserve quaternary structure and avoid introducing the potential for ionic effects.

  • If aggregation is occurring after reaction, and your protein can tolerate it, washing with 0.6 M triethylammonium bicarbonate buffer, in which Nanogold is highly soluble, can also help break up hydrophobically-induced aggregation. Preparation of triethylammonium bicarbonate buffer is described in the following reference:

    Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77-91 (1986).

  • Nonfat dried milk (up to 5%) is effective in reducing background interactions with FluoroNanogold and Nanogold probes; since background is sometimes the result of hydrophobic interactions, this may also help with aggregation resulting from similar issues. Although Nanogold is not a conventional colloidal gold, it is possible that other additives that are used to modulate hydrophobic interactions in the preparation of colloidal gold will help.

  • Addition of amphiphiles, such as benzamidine or 1,2,3-heptanetriol, may also help (0.1 to 0.2%).

Does your protein contain more than one cysteine residue, or is the labeling site formed by reduction of a disulfide bond? In this case, aggregation could be mediated by multiple thiols coordinating to the gold surface and displacing the ligands. The following suggestions may help in this case:

  • If the protein has multiple cysteines, you should adjust the ratio of Nanogold : protein in the labeling reaction to ensure 1 : 1 labeling. Use a larger excess of Nanogold than usual (10-fold excess): this will ensure that only one peptide or protein attaches to each Nanogold particle, and hence reduces the probability of forming extended cross-linked oligomers.

  • If the labeling site is generated by reduction of a disulfide bond, then you can ensure that it does not reoxidize by using rigorously degassed buffers and including 1 mM disodium EDTA in the reaction buffer.

  • Addition of 10 mM N-ethylmaleimide (NEM) once reaction is complete will cap any remaining exposed thiols and prevent them from interacting with Nanogold particles, especially when you concentrate the reaction mixture for purification and the reagents are forced into close proximity.

More information:

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Nano-W Helps Improve Antiviral Targeting

Negative stains are used to define the edges of particulate or suspended specimens with low contrast, such as protein complexes or viruses, for high-resolution electron microscopy. They are particularly important for structural studies of viruses and other macromolecular protein assemblies with a defined assembly pattern, where visualization of the entire structure and its orientation is required, such as for image analysis, rather than the localization of a specific site by gold labeling.

Negative stains are amorphous, since crystallization can obscure features of interest. When used with ultrastructural gold labeling using smaller gold particles such as Nanogold®, it is helpful if the stain is not too electron-dense, so that contrast between the gold particles and their environment is preserved. Nanoprobes offers two novel negative staining reagents, NanoVan and Nano-W. These are based on vanadium and tungsten respectively, and enable negative staining with a range of different densities. NanoVan is recommended for use with Nanogold because the lower atomic number of vanadium means that the stain is less electron-dense than heavy metal-based stains such as uranyl acetate or lead citrate, and allows easier visualization of the Nanogold particles. It is very fine-grained and highly amorphous, and has been used for a number of high-resolution STEM and TEM studies of virus and protein ultrastructure. Nano-W gives a more dense stain, and is more suited to use with larger gold labels.

Advantages of these reagents:

  • NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give any desired intermediate stain density.
  • Near-neutral pH results in better ultrastructural preservation.
  • NanoVan is less susceptible to electron beam damage than uranyl acetate.
  • Fine grain allows high imaging resolution.

[Negative Staining - Principle and Examples (41k)]

Schematic showing how negative stains work (left) and high-resolution electron micrographs obtained using a scanning transmission electron microscope. (a) Tobacco Mosaic Virus (TMV) negatively stained with 2 % uranyl acetate; (b) TMV stained with 1 % methylamine vanadate (NanoVan); both samples imaged with a dose of 104 eI/nm2. Original full width 128 nm for each image. (c) Side view of groEL (large arrow) labeled with 1.4 nm gold cluster (Nanogold, small arrow) imaged in methylamine vanadate. Note clear visibility of subunit structure and gold cluster. Full width 128 nm. Specimen kindly provided by A. Horwich, Yale University.

Bultmann and colleagues report, in a recent paper in Antimicrobial Agents and Chemotherapy, the development of improved viral inhibitors based on the TAT peptide with an added C-terminal cysteine. Peptides containing the protein transduction domain (PTD) of the human immunodeficiency virus tat protein (GRKKRRQRRR) are known to be effective inhibitors of herpes simplex virus type 1 (HSV-1) entry. The authors now show that the addition of a single cysteine residue to the C terminus of the TAT PTD (TAT-C peptide) improves antiviral activity against HSV-1 and HSV-2. Electron microscopy using negative staining with Nano-W was used to show the degradation of the viral envelope upon treatment with the modified peptide; membrane degradation was clearly visualized in comparison with a control with an intact viral envelope.

For negative stain electron microscopy, virus (5 µL of 1.0 x 1010 PFU/ml HSV-1 KOS) was adsorbed to ionized Pioloform-coated grids for 5 minutes at room temperature. The grids were then rinsed with serum-free DMEM and treated with 11.4 mM TAT-C- or n50,51 TAT-C- in serum-free DMEM or with peptide-free medium for 30 minutes at 37°C. The grids were rinsed again with serum-free DMEM, exposed to 1% OsO4 in tap water, rinsed twice with distilled water, and then negatively stained with Nano-W. Excess stain was removed, and grids were air dried. Some grids with mock-treated control virus were exposed for 1 to 2 minutes at room temperature to 2% Triton X-100 in 250 mM NaCl and 20 mM Tris-HCl (pH 8.0). Images were taken at a magnification of 100,000 in a JEOL 100CX electron microscope.

The principle effect of adding the cysteine was to enable the peptide to inactivate virions and to induce a state of resistance to infection in cells pretreated with peptide. The TAT-C peptide acted extracellularly, immediately blocked entry of adsorbed virus, prevented VP16 translocation to the nucleus, and blocked syncytium formation and cell-cell spread. Thus, TAT-C peptides are fusion inhibitors. The induction of the resistance of cells to infection was rapid, recovered with a half-life of 5 to 6 h, and could be reinduced by peptide treatment. TAT-C bound to heparan sulfate but was a poor competitor for viral attachment. The antiviral activity depended on the net positive charge of the peptide but not on chirality, and a free sulfhydryl group was not essential for antiviral activity because TAT-C dimers were at least as effective as monomers. The unique combination of antiviral activities and low toxicity combine to make TAT-C a strong candidate for further development as a drug to block HSV infection.


  • Bultmann, H.; Teuton, J., and Brandt, C. R.: Addition of a C-Terminal Cysteine Improves the Anti-Herpes Simplex Virus Activity of a Peptide Containing the Human Immunodeficiency Virus Type 1 TAT Protein Transduction Domain. Antimicrob. Agents Chemother., 51 1596-1607 (2007).

More information:

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Enhanced Plasmid DNA Transfection with Positively Charged Nanogold®

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. We have previously showed that DNA efficiently binds to Positively Charged Nanogold, and postulated this as a method for the construction of DNA nanowires. Now, in a study described in Biochimica et Biophysica Acta, Noh and group report an enhancement of the transfection efficiency of plasmid DNA through the use of Positively Charged Nanogold nanoparticles (PGN).

Plasmid DNA encoding for murine IL-2 under the control of the cytomegalovirus promoter (pVAXmIL-2) was constructed via the insertion of the 529-bp mIL-2 gene from pCIneoIL-2 gene into the pVAX1 expression vector, using NheI and BamHI restriction enzymes. For quantitative PCR, a pVAXmIL-2 deletion mutant was constructed via the insertion of a partial fragment of the mIL-2 gene (356 bp) into the pVAX1 expression vector, using NheI to HindI Plasmid DNA was purified from E. coli using a Midi kit from Qiagen. Positively Charged Nanogold (PGN), 1.4 nm in diameter with approximately 6 primary amine groups, was suspended in distilled ultrapure water to a concentration of 0.1 nmol/µL. To avoid thiol effects, exposure of Positively Charged Nanogold to thiols during the preparation of complexes was minimized. Various quantities of PGN solution (0.1 nmol/µL) were added to plasmid DNA solution (1 µg of pVAX-mIL-2 in 8 µL distilled ultrapure water) and immediately vortexed, briefly spun down, and maintained for 15 minutes at 4°C. TEM was employed to visualize the size and complexation of the PGN and DNA complexes. Formvar-coated grids (300 mesh) were utilized in all experiments. For the TEM observation of the PGN/DNA complexes, 2 µL of the solution in ultrapure distilled water was maintained on a grid for 30 min. Excess solution was then blotted off using filter paper, and the grid was air-dried. In the case of the uncomplexed PGN solution (0.1 nmol/µL), 2 µL of the solution in water was maintained on a grid for 30 min. For fresh complexes, complexation was accomplished via addition just prior to TEM analysis, and visualized under a LEO-912AB OMEGA transmission electron microscope.

Plasmid DNA encoding for murine interleukin-2 (pVAXmIL-2) was complexed with Positively Charged Nanogold (PGN) at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and the plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did polyethylenimines (PEI) of different molecular weights, such as PEI25K (MW 25 kDa) and PEI2K (MW 2 kDa). PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than was seen with PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than was seen in association with the PEI25K/DNA and PEI2K/DNA complexes, respectively. Following intramuscular administration, PGN/DNA complexes showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. Moreover, the PGN/DNA complexes showed higher cell viability than other cationic nonviral vectors. Collectively, the results of this study suggest that the PGN/DNA complexes may harbor the potential for development into efficient and safe gene delivery vehicles.


  • Noh, S. M.; Kim, W. K.; Kim, S. J.; Kim, J. M.; Baek, K. H., and Oh, Y. K.: Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles. Biochim. Biophys. Acta, 1770, 747-752 (2007).


More information:

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Going to the Venture Forum? See You There!

Nanoprobes will be presenting at the Venture Forum in San Francisco this Tuesday and Wednesday, May 1 and 2, 2007. The Venture Forum is the largest - and longest running - showcase of early stage innovation and entrepreneurship on the planet, and an ideal venue for investors and technologies to connect.

Nanoprobes will be presenting the use of Nanogold as a quencher for Molecular beacons, a topic that has been discussed recently in this newsletter. However, we have a wide-ranging research program based on the application of metal nanoparticle technology to solve biomedical problems, including the use of gold nanoparticles as improved X-ray contrast agents and as enhancers for radiation therapy, as well as the development of novel biological detection technologies. We will be presenting from 11:38 to 11:50 AM in Atrium 3, located on the Atrium Level at the San Francisco Marriott on Wednesday, May 2.

We will also be at Booth 102, located in the Golden Gate Hall of the San Francisco Marriott, on both days of the Venture Forum. Please bring any questions to the booth - you can learn more about all our technologies and business there.

More information:

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Other Recent Publications

The antibacterial effects of silver nanoparticles are well-established; however, a recent paper by Grace and Pandian in Colloids and Surfaces A: Physicochemical Engineering Aspects relates the antibacterial properties of aminoglycosidic antibiotic-protected gold nanoparticles. Gold nanoparticles were used as a probe for the detection of various aminoglycosidic antibiotics like streptomycin, gentamycin and neomycin. The drug-coated nanoparticles were prepared by treatment of 0.1 mM citrate-stabilized gold nanoparticles (10 cm3 of 0.5 mM Au(0) diluted to 50 mL) with 5 cm3 of 3 mM drugs in water and stirring for 2 hours. The nature of interaction between these drugs and gold nanoparticles has been probed by various analytical techniques like UVvisible, FT-IR, and TEM; in addition, the efficacy of the nanoparticle-drug conjugates were compared with those of the unbound drugs in bacterial cultures. These studies could be used for the determination of drugs in drug formulations. Furthermore, the in vitro antibacterial activities of drug-capped gold nanoparticles was found to be higher than those of the pure drugs against strains of Gram positive and Gram negative organisms (Staphylococcus aureus, Micrococcus luteus, E. Coli and Pseudomonas aeruginosa). These results suggest that gold nanoparticles could act as an effective drug delivery system.


  • Grace, A. N., and Pandian, K.: Antibacterial efficacy of aminoglycosidic antibiotics protected gold nanoparticles A brief study. Colloids and Surfaces A: Physicochem. Eng. Aspects, 297, 63-70 (2007).
Meanwhile, in Talanta, Hou and group report an immunoresonance scattering spectral assay for trace prealbumin. A gold-labeled probe for trace prealbumin (PA) was prepared by using gold nanoparticles of 9.0 nm diameter to label goat anti-human prealbumin polyclonal antibody. An immunoreaction between the gold-labeled antibody and prealbumin was conducted in sodium phosphate buffer solution at pH 7.6. In the presence of polyethylene glycol PEG-10000, the labeled gold nanoparticles were released and aggregated, causing the resonance scattering intensity (IRS) at 580 nm to enhance greatly. The deltaIRS is proportional to the prealbumin concentration in the range from 16.67 to 666.67 ng/mL, with a detection limit of 4.1 ng/mL. This simple, sensitive and selective assay was applied to determination of prealbumin in human plasma, with satisfactory results. However, we do wish they would not use our Nanogold® trademark for colloidal gold.


  • Hou, M.; Sun, S., and Jiang, Z.: A new and selective and sensitive nanogold-labeled immunoresoance scattering spectral assay for trace prealbumin. Talanta, 72, 463-467 (2007).

The applications of Nanogold® in neuroscience just keep going and going. In the latest publication in this field in the Journal of Neuroscience, Suzuki and co-workers use Nanogold immunolabeling with HQ Silver enhancement to demonstrate that cadherin-8 (cad8) is essential for establishing the physiological coupling between cold-sensitive sensory neurons and their target DH neurons. Classic cadherins, comprising multiple subtypes, mediate selective cell cell adhesion based on their subtype-specific binding nature. Each subtype in the brain is expressed by restricted groups of functionally connected nuclei and laminas. However, whether each subtype has any specific role in neural circuitry remains largely unknown. Cad8 was expressed by a subset of neurons in the dorsal horn (DH) of the spinal cord, as well as by a small number of neurons in the dorsal root ganglia (DRGs); the majority of cad8-positiveDRGneurons coexpressed cold temperature/menthol receptor (TRPM8). The authors generated cad8 knock-out mice and analyzed lacZ markers expressed by the targeted cad8 locus using heterozygous mice. Four ~8-week old mice were deeply anesthetized with pentbarbital and perfused through the left ventricle with a fixative containing 4% formaldehyde (detection of cad8). Segments of the spinal cord at the lumbar enlargement were dissect out and sliced transversely at a 70 µm thickness. For the detection of cad8, spinal cord slices were incubated with CAD81 for 3 d at 4°C, subsequently overnight with Nanogold anti-mouse IgG (1:100), and then enhanced with HQ Silver. Stained slices were treated with 0.5% OsO4 for 30 minutes and stained en bloc with 4% uranyl acetate in 50% ethanol. Slices were dehydrated and embedded in Epon 812. Parasagittal ultrathin sections were cut at 70-80 nm using an ultramicrotome, stained with 4% uranyl acetate in 50% ethanol and Leinolds lead citrate, and observed with an electron microscope. LacZ/cad8-expressing sensory neurons and DH neurons were connected together, and cad8 protein was localized around the synaptic junctions formed between them. This relation was, however, not disrupted in cad8-/- mice. Whole-cell patch-clamp recordings from DH neurons in spinal cord slices, in combination with menthol stimulation as a tool to excite central terminals of primary afferents expressing TRPM8, resulted in LacZ-expressing DH neurons exhibiting fast and slow miniature EPSCs. Menthol selectively increased the frequency of the slow mEPSCs in cad8+/- slices, but this effect was abolished in cad8-/- slices. The cad8-/- mice also showed a reduced sensitivity to cold temperature. These results show that cad8 is required in order to establish physiological coupling between cold-sensitive sensory neurons and their target DH neurons.


  • Suzuki, S. C.; Furue, H.; Koga, K.; Jiang, N.; Nohmi, M.; Shimazaki, Y.; Katoh-Fukui, Y.; Yokoyama, M.; Yoshimura, M., and Takeichi, M.: Cadherin-8 is required for the first relay synapses to receive functional inputs from primary sensory afferents for cold sensation. J. Neurosci., 27, 3466-3476 (2007).

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