Updated: January 7, 2005

N A N O P R O B E S     E - N E W S

Vol. 6, No. 1          January 7, 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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Nanowires from Nanogold®

Nanowires are an important goal in nanotechnology, since they are potential components of nanoscale electrical circuits and conductive detectors and other devices. However, making nanowires and controlling their properties is a challenge, and different groups have used different experimental approaches.

The use of linear, or linearly assembled, biopolymers as templates for the deposition of metal nanoparticles or metallographic deposition has produced several very promising results. We have tried this ourselves, using Positively Charged Nanogold® to decorate DNA for STEM observation. The group of Weizmann, Patolsky and Willner have used a number of different biomolecules as templates, including G-actin labeled with Mono-Sulfo-NHS-Nanogold®, and polylysine-templated oligonucleotides labeled using Mono-Sulfo-NHS-Nanogold-conjugated amino-psoralen, a photoreactive DNA intercalator.

Most recently, they have used single and double stranded DNA as templates for the attachment of Nanogold particles, which were then gold enhanced to produce conductive nanowires. DNA has important advantages as a nanowire template:

  • Oligonucleotide shape, length, and composition can be controlled synthetically.
  • Enzymes can be used to elongate, cut or replicate DNA.
  • Cation metal species can be bound to generate catalytic centers for autometallography.
  • Sequence-specific binding proteins can shield domains for creating patterned metal regions.
  • The ends can be functionalized and used to hybridize and connect to other wires or terminals.

    In this work, they use cancer cells to produce the DNA, thus circumventing the limits that oligonucleotide synthesis places on the length of the wires that may be produced. Telomerase from HeLa cancer cells was used to generate repeated DNA sequences, and these were then labeled using two methods.

    [Nanogold nanowire formation schematic] (51k)]

    Two approaches for the preparation of Nanogold-decorated nanowires using DNA.

    In the first, shown in Scheme 1, the authors used Mono-Sulfo-NHS-Nanogold to label a 12-mer oligonucleotide complementary to the telomer, which was then hybridized with the telomerase-produced DNA strand, thus spacing the Nanogold particles at short intervals along the chain.

    In the second approach, shown in Scheme 2, enzymatic telomer generation was conducted using a 1 : 10 mixture of unmodified dUTP and the amino-modified form, 5-[3-Aminoallyl]-2'-deoxyuridine 5'-triphosphate (amino-dUTP); the resulting amino-fuctionalized oligonucleotide was then gold labeled with excess Mono-Sulfo-NHS-Nanogold. The metal nanoparticles were then enlarged using a catalytic gold-based autometallography procedure similar to gold enhancement, enlarging the particles until AFM and TEM measurements indicated that they touched. Using this method, nanowires up to 3.5 microns in length could be prepared.


    • Weizmann, Y.; Patolsky, F.; Popov, I., and Willner, I.: Telomerase-Generated Templates for the Growing of Metal Nanowires. Nano Lett., 4, 787-792 (2004).

    • Patolsky, F.; Weizmann, Y., and Willner I.: Actin-based metallic nanowires as bio-nanotransporters. Nat. Mater., 3 692-695 (2004).

    • Patolsky, F.; Weizmann, Y.; Lioubashevski, O., and Willner, I.: Au-nanoparticle nanowires based on DNA and polylysine templates. Angew. Chem. Int. Ed. Engl., 41, 2323-2327 (2002).

    An alternative approach to nanowire formation is to construct from the bottom up, using conductive units that assemble to form wires. A recent example was described by Hassenkam, who used dodecane-thiol-stabilized gold nanoparticles to build a pseudo one-dimensional molecular electronic network consisting of segments of gold nanowires separated by 1-3 nm wide gaps, which were connected by oligo (phenenylene-vinylene)s (OPVs) 1.3 nm - 1.9 nm in length.

    The molecular electronic network was formed by spreading a mixture of lipid surfactant dipalmitoylphosphatidylcholine (DPPC) and 0.44 molpercent alkylthiol-protected gold particles (10 nm diameter) nanoparticles on a water surface. The Langmuir monolayer was compressed to 30-40 mN/m then transferred horizontally (Langmuir-Schäfer) to a solid support with a preformed network of gold electrodes, prepared by UV/e-beam lithography. Network conductivity, measured by AFM and by an underlying network of gold electrodes upon which the wire segments were deposited, was found to increase by 2-3 orders of magnitude with increasing covalent contact between OPV molecules and electrodes.

    The electronic properties of the networks were characterized in three situations: (i) where only lipid molecules reside in the gap between gold wire segments; (ii) where OPV molecules too short to bridge the gap reside there, or (iii) with OPV molecules long enough to bridge the gap.


    Hassenkam, T.; Moth-Poulsen, K.; Stuhr-Hansen, N.; Nørgaard, K.; Kabir, M. S., and Bjørnholm, T.: Self-Assembly and Conductive Properties of Molecularly Linked Gold Nanowires. Nano Lett., 4, 19-22 (2004).

    More information:

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    Just What is Nanogold®, Anyway?

    You never get tired of asking, and we never get tired of telling...but seriously, we are often asked what the exactly is Nanogold® - its chemical structure, how it is conjugated, and how long is the conjugation link. Here's what we know...

    What is the structure of Nanogold?

    The short answer is, we don't know. The only way to find out is to determine its crystal structure, and to date neither we nor anyone else have been able to prepare single crystals, although Nanogold occasionally shows the ability for supramolecular organization to form protocrystalline arrays.

    The best information suggests that Nanogold is a cluster compound of gold whose core contains between 55 and 75 gold atoms. Chemically, it is very similar to the smaller undecagold ([Au11]) cluster compound: it is stabilized and coordinated by the same ligands - tris (aryl) phosphines and halides or pseudohalides, which bind to surface atoms to protect and solubilize the cluster. Nanogold is thought to be a coordination compound, not a colloidal particle: its UV-visible spectrum does not show the plasmon resonance characteristic of colloidal gold. These coordinated ligands protect and stabilize the gold core more effectively than adsorbed proteins stabilize colloidal gold, and therefore Nanogold reagents and conjugates are more stable, dissociate less readily, tolerate a wider range of buffers and pH conditions, and have a longer shelf life than colloidal gold conjugates.

    For comparison, the structure of undecagold, including the attachment of the cross-linking arm, is shown on our web site. The crystal structure of undecagold has been determined, and therefore the coordination geometry of the different ligands to the surface gold atoms has been confirmed.


    Bellon, P.; Manassero, M., and Sansoni, M.: Crystal and molecular structure of tri-iodoheptakis(tri-p-fluorophenylphosphine) undecagold. J. Chem. Soc. Dalton Trans., 1481-1487 (1972).

    How is the conjugated biomolecule attached? How long is the cross-linking arm?

    Cross-linking occurs through a reactive moiety, which is incorporated as a substituent in one of the coordinated tris (aryl) phosphine ligands. The gold particle is initially prepared with a carboxypropylamino- substituent; this is then converted to either a maleimide, or to a Sulfo-succinimidyl ester. This gives the cross-link structures and lengths shown below:


    • Structure: [Au]-P-[aryl]-C(O)-NH-CH2-CH2-CH2-N-[maleimide]
    • Estimated link length: 0.6 nm.


    • Structure: [Au]-P-[aryl]-C(O)-NH-CH2-CH2-CH2-NH-C(O)-CH2-CH2-CH2-CH2-C(O)-O-[Sulfo-NHS]
    • Estimated link length: 0.9 nm.

    It should be noted that because of their very strong affinity for gold, thiol compounds are capable of displacing the coordinated phosphine ligands upon exposure to concentrations greater than about 1 mM or for times longer than about 10 minutes. Therefore, we advise against using Nanogold conjugates in systems known to contain free thiols. Thiols may be blocked before Nanogold application by treatment with N-ethylmaleimide.

    How many actual Nanogold particles are there in your products?

    Our standard packaging of 30 nmol and 6 nmol of Nanogold labeling reagents contain the following numbers of particles:

    30 nmol:

    Number of Nanogold particles

    = 30 x 10-9 x Avogadro's number

    = 30 x 10-9 x 6.023 x 1023

    = 1.81 x 1016

    6 nmol:

    Number of Nanogold particles

    = 6 x 10-9 x Avogadro's number

    = 6 x 10-9 x 6.023 x 1023

    = 3.61 x 1015

    Our 10 nmol packaging of NTA-Ni(II)-Nanogold contains 6.02 x 1015, as do our 10 nmol packaging of undecagold labeling reagents; our 50 nmol vials of undecagold reagents contain about 3.01 x 1016 undecagold particles.

    More information:

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    Gold Surfaces Change Reduction Potentials: Catalysis

    Gold nanoparticles catalyze the reduction of metal from solution around them, and the deposition of silver, gold and other ions from solution to enlarge the nanoparticles. This process is important both for the formation of metal nanoparticles - its mechanism determines their final size and size distribution - and for autometallography, or the catalytic enlargement of pre-existing nanoparticles. But why and how does this happen?

    It may be inferred that the reduction potential is favorably changed at the metal surface to accentuate the reduction reaction for ions close to the surface. Now, Vesper, Mirkin and co-workers have measured this change using redox potential measurements made with molecules containing a specific redox site coordinated to a gold surface in two tightly-packed and constrained geometries, each of which positions the redox site at a different height above the gold surface. As expected, two different redox potentials were measured, and the redox potential could be correlated with distance from the metal surface.

    [porphyrazine derivative structures] (16k)]

    Structure of porphyrazine derivatives (1)designed to form vertical self-assembled monolayers (SAMs) on gold surfaces, and (2) designed to lay flat on surfaces.

    Redox measurements were made using the two novel porphyrazine derivatives shown above, which are designed to position themselves quite differently when attached to a surface: (1) which has a single set of thiol "legs" and self-assembles in a vertical geometry, and (2) which has two opposing sets and therefore lies horizontally along the surface. Both exhibit similar reduction potentials in solution. Monolayers of each were prepared on freshly evaporated gold substrates by incubating in a CH2Cl2 solution containing 1 mM porphyrazine for 24 hours. Substrates were then rinsed with CH2Cl2 three times and ethanol three times to remove any physisorbed material and dried under a stream of N2. The monolayer-functionalized gold substrates were placed into an electrochemical cell and used as the working electrode in a three-electrode configuration with a Pt wire counter electrode: redox potentials were referenced versus a Ag/AgCl (3 M NaCl) electrode and reported versus a ferrocenium/ferrocene couple, with aqueous NaPF6 (0.5 M) and NaClO4 (1.0 M) as the supporting electrolyte. The geometries were verified using AFM.

    When adsorbed on gold, the first ring-reduction potential of (1) shifted to less negative voltages by 0.43 V, whereas that of (2), whose central ring lies closer to the surface, shifted by 0.80 V. The basis for the change in potential was thought to be dipole formation: the applied potential needed to oxidize or reduce molecules in solution reflects in part the energy needed to stabilize more highly charged species (ions versus uncharged species). If the molecules are adsorbed on a metal, the formation of a mirror-image charge dipole on the gold surface reduces the energetic expense of charged ion solvation. As expected, the contribution of this effect decreases as the redox center is moved away from the gold surface.


    Vesper, B. J.; Salaita, K.; Zong, H.; Mirkin, C. A.; Barrett, A. G., and Hoffman, B. M.: Surface-bound porphyrazines: controlling reduction potentials of self-assembled monolayers through molecular proximity/orientation to a metal surface. J. Amer. Chem. Soc., 126, 16653-16658 (2004).

    Apart from autometallography, can gold nanoparticles perform other catalytic tranformations? Tsunoyama and co-workers confirm that they can with their recent report that gold nanoparticles can catalyze carbon-carbon bond formation. Poly(N-vinyl-2-pyrrolidone)(PVP)-stabilized gold nanoparticles (<2 nm diameter), were prepared by sodium borohydride reduction of tetrachloroaurate in the presence of PVP, then used for the homocoupling of phenylboronic acid to give biphenyl as the major product and phenol as a minor product.

    To determine catalytic activity, phenylboronic acid (54.8 mg, 0.45 mmol), K2CO3 (186.6 mg, 1.35 mmol), and H2O (5 mL) were placed in a 100-mL Erlenmeyer flask and aqueous PVP-gold nanoparticles (0.45mM, 10 mL, 1 at. %) added. The reaction mixture was stirred vigorously at room temperature under aerobic conditions for 24 hours, quenched with 1 M HCl, and organic products extracted with ethyl acetate. The organic layer was dried over Na2SO4 and evaporated in vacuo, and products analyzed by 1H NMR. The concentration of oxygen dissolved in water was measured by using a DO electrode.

    Yields were severely reduced under anaerobic conditions, implying that molecular oxygen plays a role similar to that in catalytic Pd(II) species. The particles were enlarged during catalytic reaction to 3 - 3.5 nm in diameter, but were found to retain catalytic activity. Based on the requirement for molecular oxygen and the influence of steric effects on product yield, a mechanism was inferred in which coordination of molecular oxygen to the gold surface is followed by transmetallation and insertion of the oxygen into the B-C(phenyl) bond of phenylboronic acid, transmetallation of a second phenylboronic acid, then elimination of [(OH)2(BO)2] and 2OH- followed by reductive elimination of biphenyl.


    Tsunoyama, H.; Sakurai, H.; Ichikuni, N.; Negishi, Y., and Tsukuda, T.: Colloidal gold nanoparticles as catalyst for carbon-carbon bond formation: application to aerobic homocoupling of phenylboronic acid in water. Langmuir, 20, 11293-11296 (2004).

    More information:

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    Nanogold®-Silver and Enzyme Double Labels Neuronal Connections

    In order to study connections of GABAergic neurons in the supratrigeminal region (Vsup) and the intertrigeminal region (Vint) regions of the rat brain, Li and co-workers combined double imunohistochemistry, retrograde and anteretrograde tracing in the adult heterozygous GAD67-GFP knock-in mouse, in which green fluorescence protein (GFP) is expressed in GABAergic neurons under the control of the endogenous GAD (GAD67) gene promoter, with an electron microscopic double labeling method in which two targets were labeled with silver-enhanced Nanogold® and enzymatically deposited diaminobenzidine (DAB) respectively.

    For the electron microscopic studies, six anesthetized mice were tereotaxically injected with a volume of 0.2 ml of 10 % (w/v) biotinylated dextran amine (BDA) in 0.05 M phosphate-buffered saline (PBS) (pH 7.3). After the injection, the mice were allowed to survive for 45 days; they were then deeply anesthetized and perfused transcardially with 100 ml of 0.1 M phosphate buffer (PB) (pH 7.4) containing 4 % (w/v) paraformaldehyde, 0.1 % (w/v) glutaraldehyde and 15 % (v/v)-saturated picric acid. The lower brainstems were cut serially into 50-micron cross-sections on a vibratome; sections containing the Vsup and/or Vint were placed for 1 h in 0.05 M PB (pH 7.4) with 25% (w/v) sucrose and 10% (v/v) glycerol, then freeze-thawed with liquid nitrogen for enhancement of antibody penetration in the immunohistochemical reaction. Non-specific interactions were then blocked by further incubation at room temperature for 1 h with 50 mM TrisHCl-buffered saline (TBS; pH 7.4) containing 20 % (v/v) normal goat serum.

    Green Fluorescent Protein (GFP) and biotinylated dextran amine (DDA) were labeled, respectively, by the immunogoldsilver method and the immunoperoxidase method. For labeling GFP with immunogoldsilver, sections were first incubated with 0.8 mg/ml anti-GFP rabbit IgG at room temperature for 24 hin an incubation medium comprising Tris-buffered 0.9 % saline (TBS, pH 7.4) containing 2 % normal goat serum (TBS-G). After washing in TBS, the sections were further incubated at room temperature overnight with Nanogold goat anti-rabbit IgG antibody, diluted at 1:100 with the incubation medium (prepared using TBS-G). The sections were post-fixed with 1 % (w/v) glutaraldehyde in 0.1 M PB (pH 7.4) for 10 minutes and washed in distilled water. Subsequently, silver enhancement was done in the dark with HQ Silver.

    Following silver enhancement, the sections were incubated at room temperature for 3 hours with the ABC-Elite Kit (Vector), diluted 1:50 with 0.05 M TBS. For labeling BDA by the immunoperoxidase method, the sections were further incubated with 0.05 M TrisHCl (pH 7.6) containing 0.02 % (w/v) DAB and 0.003 % (v/v) H2O2 at room temperature for 2030 min. Then the sections were placed in 0.1 M PB (pH 7.4) containing 1 % (w/v) OsO4 for 1 hour. Subsequently, the sections were counterstained with 1% (w/v) uranyl acetate in 70 % ethanol for 1 hour. After dehydration, the sections were mounted on silicon-coated glass slides and flat-embedded in epoxy resin. The flat-embedded sections were examined under a dissection microscope and small pieces of the Vsup or Vint were cut out with fresh razor blades. The selected tissue pieces were cut into 50-nm-thick ultrathin sections (silver sections) with a diamond knife mounted on a ultramicrotome.

    The ultrathin sections, mounted on single-slot grids coated with pioloform membrane, were then examined in the electron microscope. BDA-labeled axon terminals were filled with round synaptic vesicles, and were in asymmetric synaptic contact with unlabeled dendritic profiles, or with silver grain-containing dendritic profiles. On the other hand, axon terminals, which are labeled with silver grains, contained pleomorphic synaptic vesicles and were in symmetric synaptic with unlabeled dendritic profiles.

    Correlation of the GFP, immunofluorescence, and tracing data with the electron microscope labeling indicated that the Vsup and Vint of the mouse contained GABAergic neurons, which received projection fibers from the marginal layer of the nucleus of the spinal tract of the trigeminal nerve (Vc) on the ipsilateral side, and sent axons to the Vm on the contralateral side. Some of these GABAergic neurons may represent Vm-premotor neurons that receive nociceptive input from the Vc to elicit the jaw-opening reflex by inhibiting jaw-closing Vm-motoneurons.


    Li, J. L.; Wu, S. X.; Tomioka, R.; Okamoto, K.; Nakamura, K, Kaneko, T., and Mizuno, N.: Efferent and afferent connections of GABAergic neurons in the supratrigeminal and the intertrigeminal regions An immunohistochemical tract-tracing study in the GAD67-GFP knock-in mouse. Neurosci. Res., 51, 81-91 (2005).

    More information:

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    Other Recent Publications

    Lee, Mirkin and group describe an nanometer-scale antibody array-based assay to determine the presence of the human immunodeficiency virus type 1 (HIV-1) in blood samples. Dip-pen nanolithography was used to generate nanoscale patterns of antibodies against the HIV-1 p24 antigen tethered to a gold surface; HIV-1 p24 antigen in plasma, obtained directly from HIV-1-infected patients, was allowed to react with the antibody, and the bound protein was then detected by the binding of a 20 nm gold antibody probe for signal enhancement. The nanoarray features in the three-component sandwich assay were confirmed by atomic force microscopy (AFM). Measurable amounts of HIV-1 p24 antigen were demonstrated in plasma obtained from men with less than 50 copies of RNA per ml of plasma (corresponding to 0.025 pg per ml): this sensitivity indicates that the nanoarray-based assay can exceed the limit of detection of conventional enzyme-linked immunosorbent assay (ELISA)-based immunoassays (5 pg per ml of plasma) by a factor of 1000-fold or more.


    Lee, K.-B.; Kim, E.-Y.; Mirkin, C. A., and Wolinsky, S. M.: The Use of Nanoarrays for Highly Sensitive and Selective Detection of Human Immunodeficiency Virus Type 1 in Plasma. Nano Lett., 4, 1869-1872 (2004).

    If you are interested in magnetic nanoparticles, Lyon and co-workers have prepared 60 nm gold-coated iron oxide nanoparticles, using a mixture of tetrachloraurate and hydroxylamine hydrochloride to deposit gold onto the surface of 9 nm either alpha-Fe2O3 or partially oxidized Fe3O4 particles. The morphology and optical properties of the core/shell particles are dependent on the quantity of deposited gold; the gold-coated particles exhibit a surface plasmon resonance peak that blue-shifts from 570 to 525 nm with increasing gold deposition. The magnetic properties, however, remain largely independent of Au addition: SQUID magnetometry indicated that particle magnetic properties are not affected by the moderately thick gold shell.


    Lyon, J. L.; Fleming, D. A.; Stone, M. B.; Schiffer, P., and Williams. M. E.: Synthesis of Fe Oxide Core/Au Shell Nanoparticles by Iterative Hydroxylamine Seeding. Nano Lett., 4, 719-723 (2004).

    Namimatsu and co-workers report a universal antigen retrieval method in the latest issue of the Journal of Histochemistry and Cytochemistry. They find that treatment with citraconic anhydride effectively reverses the effects of formalin fixation for a wide range of samples. The method is straightforward: deparaffinized sections on slides in stainless steel staining racks were placed in an electric kitchen pot (Matsushita Electric Co.) filled with about 800 ml of 0.05 % citraconic anhydride solution, pH 7.4, so the solution covers the racks, and the pot set to "keep warm" temperature mode (98°C) for 45 minutes.


    Namimatsu, S.; Ghazizadeh, M., and Sugisaki, Y.: Reversing the effects of formalin fixation with citraconic anhydride and heat: A universal antigen retrieval method. J. Histochem. Cytochem., 533-11 (2005).

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