N A N O P R O B E S     E - N E W S

Vol. 7, No. 3          March 15, 2006

Updated: March 15, 2006

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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Gold Nanoparticles as X-Ray Contrast Agents

Triiodobenzene derivatives have been the chemical platform of choice for X-ray contrast agents for more than 25 years for applications such as angiography. However, they have serious limitations for many applications: short imaging times, the need for catheterization in many cases, high viscosities that can cause arterial damage when injected and excludes their use in smaller vessels, occasional renal toxicity, and poor contrast in large patients. As part of our research at Nanoprobes, we have investigated the potential of gold nanoparticles as improved X-ray contrast agents, and we now report that gold nanoparticles may overcome many of these limitations. Gold has higher X-ray absorption than iodine with less bone and tissue interference, thus achieving better contrast with lower X-ray dose. Because nanoparticles clear the blood more slowly than iodine agents, they permit longer imaging times.

Gold nanoparticles, determined by electron microscopy to be 1.9 nm in diameter, at a concentration of injected gold of 270 mg Au cm-3 suspended in phosphate-buffered saline at pH 7.4, were injected via the tail vein into Balb/C mice bearing EMT-6 subcutaneous mammary tumors at an injection volume of 0.01 mLg-1 mouse weight. The vascular system was imaged in planar projection over time using a using a standard mammography unit (Lorad Medical Systems). Gold biodistribution was then measured in different tissues by graphite furnace atomic absorption spectrometry. Retention in liver and spleen was low, with elimination by the kidneys.

A 5 mm tumour growing in one thigh was clearly evident from its increased vascularity and resultant higher gold content. These nanoparticles thus enable direct imaging, detection, and measurement of angiogenic and hypervascularized regions. Images taken at various times after intravenous injection show that the 1.9 nm gold nanoparticles do not concentrate in the liver and spleen, but clear through the kidneys. A closer examination of the kidneys revealed a remarkably detailed anatomical and functional display; organs such as kidneys and tumors were seen with unusual clarity and high spatial resolution, and blood vessels less than 100 mm in diameter were delineated, thus enabling in vivo vascular casting.

Toxicity and clearance are critical issues for clinical use of imaging agents. Mice intravenously injected with the gold nanoparticles at 2.7 g Au kg-1 survived over one year without signs of illness. The LD50 for this material was found to be approximately 3.2 g Au kg-1. In a 30-day toxicity study using 60 mice, intravenous injection of the gold nanoparticles (initially, 10 mg Au mL-1 blood) showed normal hematology and blood chemistry. Histological examination of 24 vital organs and tissues from each mouse, assayed 11 days or 30 days after injection of the nanoparticles, showed no evidence of toxicity in any animals. Quantitative pharmacokinetics using graphite furnace atomic absorption spectroscopy (Figure 4) showed that blood gold concentration decreased in a biphasic manner, with a 50% drop between 2 min and 10 min followed by a slower decrement of another 50% between 15 min and 1.4 h. The highest tissue gold concentration 15 min after injection was in the kidney, followed by tumour, liver and muscle (1.20.1%id/g). In addition, the gold nanoparticle reagent demonstrated a relatively long tumor residence time which resulted in increased contrast between tumor and non-tumor tissue: muscles and blood were almost gold-free 24 hours after injection (0.28±0.07%id/g and 0.100.01±id/g respectively), whereas at 24 hours the tumor retained 64% of its value at 15 min. The tumour : muscle gold ratio was 3.4 at 15 min post injection, improving to 9.6 at 24 h, enabling clear delineation of the tumor. In addition to imaging, higher tumor X-ray absorption due to the gold has been shown to greatly improve the efficacy of radiotherapy.

Even when concentrated, gold nanoparticle solutions were similar to water in viscosity, in sharp contrast to the high viscosity of iodine contrast media. The gold nanoparticles may be completely dried and later resuspend easily in water or aqueous buffers, such as phosphate buffered saline, pH 7.4. Solubility was found to be at least 1.5 g Au ml-1. The gold nanoparticles are stable: no changes were observed in their UV/visible spectra or aggregation was found after storage for 6 months at 4°C or 220°C.

These animal studies demonstrate the potential of gold nanoparticles as X-ray contrast agents with important physical and pharmacokinetic advantages over current agents. They appear to be non-toxic and enable higher contrast and longer imaging times than currently possible using standard iodine-based agents.


Hainfeld J. F.; Slatkin D. N.; Focella T. M., and Smilowitz H. M.: Gold nanoparticles: a new X-ray contrast agent. Br. J. Radiol., 79, 248-253 (2006).

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Background Troubleshooter: Different Probes and Enhancement Methods

Background staining - the development of signal in the absence of the specific target you wish to stain - is one of the most frequent problems with immunogold staining and autometallography. In previous issues of this newsletter, we have discussed specific methods for reducing background with different types of immunoprobes, including Nanogold and FluoroNanogold, and the different autometallographic enhancement procedures - silver enhancement and gold enhancement.

However, the best method for removing background signal depends on what is causing your background, and therefore it is worth investigating which reagent is to blame. Non-specific signal can arise from non-specific binding of an unlabeled primary antibody or probe, from a gold-labeled probe sticking to other parts of the system, or from the reaction of silver or gold enhancement reagents with other components of your specimen. A general approach which is often helpful is to remove each component in turn from your system, and compare the effect on background signal:

  • Omit the primary antibody and apply the gold- or FluoroNanogold-labeled secondary and (if you use it) silver enhance as usual. If the background disappears, it may be due to the primary antibody. In this case, reducing the concentration of primary antibody or using more thorough washing or permeabilization after incubation may help reduce background. Another potential source of background is the presence of traces of amine-reactive fixatives, such as glutaraldehyde, which can react with proteins: these should be quenched with glycine, ammonium chloride, or sodium cyanoborohydride before immunostaining.

  • Omit the gold-labeled secondary, but apply the primary and silver or gold enhancement reagents as usual. If this fixes the problem, then it may be due to non-specific binding by the gold conjugate. In this case, solutions should address the mechanisms by which gold might adsorb to specimen components:

    • 5 % nonfat dried milk has been found to be highly effective in reducing FluoroNanogold and Nanogold background. This was found to be particularly effective when mixed with and added to the specimen together with the FluoroNanogold conjugate.

    • Background binding is often attributed to hydrophobic interactions (both gold and fluorescent labels have some hydrophobicity). Adding reagents that reduce hydrophobic interactions to the wash buffer or the gold conjugate incubation buffer may reduce non-specific binding. Examples include 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling carbon dioxide into an aqueous suspension of triethylamine with stirring. 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 (1986)); 0.1 % to 1 % detergent, such as Tween-20, or Triton X-100; and 0.1 % to 0.5 % of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane.

    • Does the distribution of the binding suggest a blocking method? For example, if it occurs in thiol (cysteine)-rich regions, it may be due to thiol coordination to the gold. This may be blocked using N-ethylmaleimide. If it occurs in nuclear material, it may be due to interactions with the ionic charges of nucleic acids; increasing the ionic strength of the buffer, or changing the pH to a value at which the nucleic acids are less ionized, may help.

    • With FluoroNanogold, manual camera exposure can help in reducing fluorescence background. FluoroNanogold is frequently compared with commercially available fluorescently labeled IgG conjugates, which are larger and more highly labeled and give brighter fluorescence. Automatic exposure adjustment with FluoroNanogold-stained specimens can result in greater exposure and higher apparent backgrounds: Setting the camera exposure manually can be used to overcome this effect.

  • If neither approach reduces background, it maybe due to the autometallographic reagent - the silver or gold enhancement step. Remedies should therefore address the redox chemistry of these reagents:

    • Wash samples with a chelating agent before silver enhancement to sequester redox-active transition metals that can catalyze silver enhancement. Examples include 0.02 M sodium citrate buffer, pH 7.0 (with Danscher silver enhancer), 0.02 M sodium citrate buffer at pH 3.5 (with HQ Silver) (Reference: Powell, R. D.; Halsey, C. M.; Spector, D. L.; Kaurin, S. L.; McCann, J., and Hainfeld, J. F.: A covalent fluorescent-gold immunoprobe: simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy. J. Histochem. Cytochem., 45 947-956(1997)) or disodium EDTA (0.05 M, pH 4.6) - use as the last wash before adding the silver enhancement reagent.

    • Ensure that all samples are thoroughly washed with ultrapure or deionized water before silver enhancement to remove halide ions: these will react with any silver salts to form a precipitate, which is visible in the EM and will nucleate additional background staining.

    • Use plastic or teflonized (not metal) forceps and tools for handling specimens.

  • If all else fails, include a reaction termination or back-development step after silver or gold enhancement to remove excess silver or gold. Methods include:

    • Apply freshly prepared 1% sodium thiosulfate solution for one minute (or longer) until the excessive gold deposits are removed.

    • Treat with Lugol's iodine for 30 seconds to one minute, then remove the residual orange-brown color of the iodine using freshly prepared 1% sodium thiosulfate solution

    • Back develop with Farmer's Solution (0.3 ml 7.5% potassium ferricyanide, 1.2 ml of 20% sodium thiosulfate, 60 ml water).

More information:

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NTA-Ni(II)-Nanogold®: Role of Wzc in the Assembly of e.coli Group 1 Capsules

NTA-Ni(II)-Nanogold® has already demonstrated advantages over antibody and protein conjugates for localizing His-tagged targets, and Collins and co-workers now report, in the Journal of Biological Chemistry, that labeling has sufficient resolution to help solve the structure of the Wzc tetramer in e. coli group 1 capsules.

NTA-Ni(II)-Nanogold is much smaller than immunogold probes or other probes, and this gives it several advantages for high-resolution electron microscopic applications, as well as general labeling and detection:

  • This probe brings the gold nearer to the target, and labeling resolution is higher. As shown below, binding-site to gold distance is only about 2.5 nm with NTA-Ni(II)-Nanogold, but would be at least 6 nm with antibody-gold probes.

  • NTA-Ni(II)-Nanogold is better able to penetrate into specimens and access restricted sites within them, and can pack closer to achieve higher labeling density. It also perturbs biological structures and processes less than a larger antibody probe would.

  • Binding constants for Ni(II)-NTA are very high due to the chelate effect of multiple histidine binding and multiple Ni(II)-NTA functionalization. Dissociation constants are estimated to be between 10-7 to 10-13 M-1. For many applications, this provides binding strengths comparable to antibodies.

  • NTA-Ni(II)-Nanogold has very high solubility and stability. At 1.8 nm in size, it is readily visualized by electron microscopy yet small enough for highest resolution.

Escherichia coli cells are protected by a structural layer, the capsule, which protects the encapsulated bacteria from host immune defenses. The capsule is formed from K antigenic capsular polysaccharide: its assembly and translocation require proteins in the inner and outer membranes. The inner membrane protein, Wzc, is a tyrosine autokinase with a central role in an apparently coordinated biosynthesis and secretion process. Mutants lacking Wzc are unable to polymerize high molecular weight capsular polymers. Homologs of Wzc have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria, and therefore the structure and function of this protein are of considerable interest.

Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc, and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. The protein structure was determined through single-particle averaging electron density mapping using data obtained from cryoelectron microscopy, both with and without NTA-Ni(II)-Nanogold labeling. The open reading frame of the gene encoding E. coli Wzc with an N-terminal His tag (His6-Wzc) was cloned in the expression vector pWQ305 (pBAD24-derivative) and transformed into E. coli CWG281 cells. 10-liter agitated fermenter batches of LB medium (containing 100 micrograms/m ampicillin) were used to grow bacteria expressing His6-Wzc. After extraction and purification of the His6-Wzc, labeling with NTA-Ni(II)-Nanogold was accomplished by incubating 10 microliters of His6-Wzc (50 micrograms/ml)with 3.3 microliters (1.8 nM) of Ni-NTA-Nanogold (Nanoprobes) for 18 h at 4°C. Samples were then centrifuged at 13,000 rpm in a bench-top centrifuge for 10 minutes before cryo-negative staining. For cryo-EM, the protein was applied to glow-discharged carbon-coated copper grids (No. 400), placed shiny side down on the surface of a 3-microliter droplet of sample containing 50 micrograms/ml His6-Wzc for 2 minutes. These were then placed on a 10-microliter droplet of freshly prepared 12% (w/v) ammonium molybdate tetrahydrate (pH 6.8) containing 1% (w/v) trehalose for several seconds. After brief blotting onto double-layered Whatman 50 filter paper, data were immediately recorded using an Oxford system cryo-stage at ~100 K. Images for each oligomeric complex were recorded and processed.

Particles were interactively selected using BOXER software and contrast-normalized. The contrast transfer function (CTF) for each micrograph was determined using the program CTFIT. Corrections for amplitudes and phases for particles in each dataset were applied using a structure factor dataset to flip phases. A set of reference-free class averages were then generated with the complex orientated in multiple particle positions, and following previously described strategies, a preliminary three-dimensional model was determined from class averages that represented distinct views of the Wzc complex. The resulting averages were combined to generate the preliminary three-dimensional model, and this structure was refined with C4 symmetry, using eight rounds of iterative projection matching. The final three-dimensional volume was fully converged, based on comparison of the Fourier shell correlation of the three-dimensional models generated from each iteration, after six rounds of iterative refinement; resolution was determined by Fourier shell correlation analysis.

From single particle averaging on the cryo-negatively stained samples, the first three-dimensional structure of this type of membrane protein in its phosphorylated state was produced at about 14 resolution. Viewed from the top of the complex, the oligomer is square with a diameter of ~100 Å and can be divided into four separate densities. From the side, Wzc is ~110 high. The complex has a distinctive appearance, similar to a molar tooth: an upper "crown" region, about 55 Å high, which forms a continuous ring of density, and four unconnected "roots" (~65 high) emerging from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.

[NTA-Ni(II)-Nanogold and Wzc protein labeling (53k)]

left: Structure of Ni-NTA-Nanogold®, showing interaction with a His-tagged protein; upper image shows comparison of resolution for gold-labeled Fab' fragment with that for NTA-Ni(II)-Nanogold. Lower section shows knob protein from adenovirus cloned with 6x-His tag, labeled with Ni-NTA-Nanogold, column purified from excess gold, and viewed in the scanning transmission electron microscope (STEM) unstained (Full width approximately 245 nm). right: Binding sites of NTA-Ni(II)-Nanogold in the assembled Wzc protein tetramer, as found by cryo-EM with negative staining and image analysis of NTA-Ni(II)-Nanogold-labeled protein.

NTA-Ni(II)-Nanogold was used both as a heavy atom derivative to assist with phasing, and as a high-resolution label to determine the orientation of the complex within the membrane. Wzc particles incubated with NTA-Ni(II)-Nanogold were essentially identical to those without Nanogold, but multiple gold densities were easily observed bound to the proteins. With a smaller gold-labeled data set, a second three-dimensional structure at ~22 Å resolution was generated which showed additional bound gold density contained within the volume. Nanogold was found in two locations: on the upper half of the root regions, and at the bottom of the cavity formed by the roots underneath the crown. This second location is not physically connected to the protein envelope, and therefore may represent nonspecific binding or trapped gold particles. However, the presence of Nanogold at either location suggests that the roots contain the N terminus, and hence are in the cytoplasm, while the crown is in the periplasm. Given the location of the N terminus on a root, the alternatively flipped orientation is inconsistent with relative observed volumes of the protein domains, and therefore the root was predicted to contain the tyrosine kinase domain. Further confirmation was provided by manually fitting the structure of MinD (an ATP-binding protein) from P. horikoshii into an individual root volume: a good volume match was found between the MinD model and molecular envelope of Wzc, consistent with the experimentally determined location of the two domains - although at 14 Å resolution, the MinD model can only act as an appropriately sized globular volume, and its precise orientation within the root domain is not possible. The overall appearance further suggests this orientation: the globular, compact appearance of the roots are also as would be expected for a soluble kinase-like domain.


Collins, R. F.; Beis, K.; Clarke, B. R.; Ford, R. C.; Hulley, M.; Naismith, J. H.; and Whitfield, C.: Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules. J. Biol. Chem., 281, 2144-2150 (2006).

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Colorimetric Kinase Activity Detection by Nanoparticle Modification

The color change that occurs when gold nanoparticles aggregate has been used for a number of assays, including recently for rapid colorimetric detection of PCR products, and colorimetric detection of metal ions, particularly lead. Wang, Brust and co-workers now report, in their recent paper in the Journal of the American Chemical Society, the application of this method to detect kinase activity.

The phosphorylation of proteins by kinases has vital regulatory roles in most metabolic pathways and in cell communication, and the identification of kinases, substrates, and potential inhibitors is required to understand many fundamental biochemical processes and in drug discovery. Conventionally, this is done by radiolabeling the substrate with gamma-32P-ATP as cosubstrate. Recently, this group demonstrated that replacing the radiolabeled cosubstrate with gamma-biotin-ATP and binding of gold-labeled avidin to the resulting biotinylated substrate allows the identification of kinase substrates by resonance light scattering of the nanoparticles. Now, they report the use of the same labeling and binding strategy (substrate biotinylation and binding to avidin-modified gold nanoparticles) to develop a colorimetric assay for kinase inhibitors.

The assay utilized peptide-capped thiol-modified gold nanoparticles, in which 10% of the peptide ligands carry an extension which is a substrate for a specific kinase: this has the effect of replacing the specific substrate with gold nanoparticles. 13-nm gold nanoparticles, stabilized by a pentapeptide, were additionally modified with two further oligopeptide sequences which are the known substrates of the cAMP-dependent protein kinase A (PKA) and the calmodulin-dependent kinase II (CaM KII) respectively. Two different kinases were used to demonstrate the potential of this method to screen for inhibitors of several kinases with the same type of nanoparticles. In separate experiments, these particles were incubated with either PKA or CaM KII, both in the presence of gamma-biotin-ATP, followed by purification and addition of 13-nm avidin-modified gold nanoparticles. The same experiments were repeated in the presence of well-known kinase inhibitors, H89 for PKA and KN62 for CaM KII.2a The solutions were then analyzed by visual inspection and UV-visible spectroscopy. In the presence of an active kinase, the color of the solutions changed from red to blue after addition of the avidin-modified particles due to the well-established shift and broadening of the plasmon resonance band in gold nanoparticle aggregates; over a period of several hours complete precipitation of the particles was observed. This effect was reduced in the presence of the inhibitor, and peak position for the plasmon resonance was found to be dependent on the inhibitor concentration, allowing for estimation of the IC50 values of the inhibitors.

These reactions were all transferred to a multiwell microplate and used to evaluate the efficiencies of three different inhibitors (H89, KN62, and SB203508) for the two kinases (PKA and CaM KII) simultaneously. Inhibitor efficacy can be inferred almost immediately from the color changes within the wells: a red solution indicates enzyme inhibition, while a blue solution indicates enzymatic biotinylation of the substrate nanoparticles. Visual inspection indicates that H89 inhibits PKA but not CaM KII, KN62 inhibits CaM KII but not PKA, and SB 203580 inhibits neither of the two kinases, in accordance with the known inhibitor activities of these three compounds. This shows that the use of specifically designed, peptide-stabilized gold nanoparticles as artificial substrates for kinases provides for a very simple colorimetric protocol, based on their unique optical properties, for evaluating kinase activity and inhibition, with potential applications in drug discovery.

Development of the conjugation chemistry of gold nanoparticles is a research focus at Nanoprobes, and we provide a range of products for specific linking to any molecule with a suitable reactive group, including Monomaleimido Nanogold® for labeling molecules with thiol groups, Mono-Sulfo-NHS-Nanogold for labeling amines, and Monoamino Nanogold for use in a variety of cross-linking reactions. Our recent introduction of NTA-Ni(II)-Nanogold now provides a simple method for either detection of, or conjugation to, polyhistidine-tagged targets.


Wang, Z.; Levy, R.; Fernig, D. G., and Brust, M.: Kinase-catalyzed modification of gold nanoparticles: a new approach to colorimetric kinase activity screening. J. Amer. Chem. Soc., 128, 2214-2215 (2006).

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Nanoprobes, Incorporated Completes Major Expansion

Nanoprobes, Incorporated announces a major expansion in its office and laboratory facilities in Yaphank, NY. The company has almost doubled its laboratory and office space, and completed an extensive renovation to upgrade its facilities, research and manufacturing capabilities. Within the past 12 months, the Company has also increased its staff by more than 30%. This expansion will deepen the Companys commitment to developing novel products and technologies, while helping to provide improved support to current customers. The expanded facilities will support research projects which include novel technology for breast cancer diagnosis and characterization, and the application of metal nanoparticles as contrast agents for biomedical imaging.

Nanoprobes, Inc., founded in 1990, researches and develops novel biomedical and microscopic staining and imaging technology using metal nanoparticles and autometallography. The company is a leader in immunogold technology, with products which include the 1.4 nm Nanogold® immunoprobes and labeling reagents, FluoroNanogold combined fluorescent and gold probes, and autometallographic enhancers for ultrasensitive visualization of biological targets. The Company recently executed a licensing agreement with Ventana Medical Systems, Incorporated (VMSI) for the use of its Enzyme Metallography (EnzMet) technology as an ultrasensitive staining method for in situ hybridization and immunohistochemistry.

For more information, contact:

Victoria Kowalski
Nanoprobes, Incorporated
95 Horse Block Road
Yaphank, NY 11980-2301
                Telephone: (631) 205-9490
Fax: (631) 205-9493
E-mail: nano@nanoprobes.com
Web: www.nanoprobes.com

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Other Recent Publications

The toxicity of other types of gold nanoparticles is also of interest. [Au55], a cluster compound described in many publications, if its coordinated ligands are removed, can coordinate irreversibly in the major grooves of DNA, the height of which perfectly fits its size. The negatively charged phosphates of the DNA backbone were found to at least partially remove the original ligands with which the clusters were protected. Gold, as the most electronegative metal, prefers the negatively charged vicinity in the DNA groove to its original neutral phosphines, and also reach a polydentate ligand sphere that increases the kinetic stability by orders of magnitude. This finding prompted an investigation of the toxicity of [Au55], which they report in Small. In tests with eleven cell lines, [Au55] was shown to be most highly cytotoxic towards the metastatic melanomas MV3 and BLM: the clusters caused 100% death of the cells at a concentration of 0.4 mM, whereas for cisplatin at the same concentration, almost 90% of the cells are still viable. The effective concentration to reduce the cell number by 50% is approximately 180 times lower for [Au55] clusters than for cisplatin for the BLM melanoma cells and over 200 times lower for the MV3 cell line. While not to the same extent, several other cell lines showed enhanced sensitivity towards [Au55]. Comparison with the results found with 1.9 nm gold nanoparticles described above shows the extent to which modification of the surface functionality of the gold can affect its biological behavior and toxicity.


Tsoli, M.; Kuhn, H.; Brandau, W.; Esche, H., and Schmid, G.; Cellular Uptake and Toxicity of Au55 Clusters. Small,, 1, 841-844 (2006).

Schneider and co-workers add substantially to our knowledge of the interactions between gold nanoparticles and fluorophores in their paper in the current Nano Letters. They synthesized fluorescent core/shell nanoparticles using electrostatic layer-by-layer assembly, comprising gold cores with a diameter of 13 nm and different fluorescently labeled polymer corona layers spaced at various distances from the surface of the core metal using nonfluorescent polyelectrolyte spacer layers. Several primer layers of low molecular weight poly(allylamine hydrochloride) (PAH, MW ) 15 000 g/mol) and poly(stryrene sulfonate) (PSS, MW ) 13 400 g/mol) were adsorbed consecutively in a layer-by-layer fashion, to obtain gold nanoparticles coated with 2, 10, and 20 layers. Fluorescein isothiocyanate (FITC) and lissamine rhodamine B sulfonyl chloride (LISS) were then covalently attached to PAH to obtain PAH-FITC and PAH-LISS respectively. Only a small fraction (approximately 1%) of the available amino groups was labeled. PAH-FITC or PAH-LISS were adsorbed onto the Au(PAH/PSS)n core-shell particles, followed by the adsorption of a final PSS layer. UV-visible spectroscopy and transmission electron microscopy confirmed that the particle suspensions of fluorescently labeled core/shell nanoparticles are stable at all stages of their construction. Photophysical investigations reveal strongly distance-dependent fluorescence quenching in these particle systems. The contribution of the metal core to this quenching can be assessed precisely by comparing the fluorescence before and after gentle dissolution of the gold cores with potassium cyanide. Photophysical measurements reveal clearly that the gold nanoparticles decrease the transition probability for radiative transitions, in accord with the recent work of Dulkeith.


Schneider, G.; Decher, G.; Nerambourg, N.; Praho, R.; Werts, MH., and Blanchard-Desce, M.: Distance-Dependent Fluorescence Quenching on Gold Nanoparticles Ensheathed with Layer-by-Layer Assembled Polyelectrolytes. Nano Lett., 6, 530-536 (2006).

Pre-embedding Nanogold® publication of the month goes to Lim et al for their localization of UT-A and UT-B in rat kidneys in different stages of hydration, reported in American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. To determine the distribution of urea transporters in the rat kidney, rabbit polyclonal primary antibodies against peptides based on the rat renal urea transporter UT-A1, UT-A2, and UT-A4 (L194 and L403) (37, 51, 58); UT-A1, UT-A3, and UT-A4 (L446) (58); UT-A3 (Q2) (52); and UT-B (38, 53), the human erythrocyte urea transporter were used, with either immunoperoxidase (for light microscopy) or Nanogold (for electron microscopy) secondaries. Vibratome sections were incubated with primary antibodies overnight, washed with 0.1% BSA, 0.05% saponin, and 0.2% gelatin-PBS (solution C) and then washed with 0.8% BSA-0.1% gelatin-2 mM NaN3-PBS and 5% normal goat serum, pH 7.4 (gold buffer). Sections were then incubated overnight at 4°C in Nanogold-IgG and Fab' conjugates, washed with PBS, postfixed with 1% glutaraldehyde for 10 minutes, then enhanced for 78 min with HQ Silver, rinsed, postfixed, and embedded for EM. Comparison of labeling for dehydrated rats given minimum water, hydrated rats given 3% sucrose in water for 3 days before death, and control rats given free access to water, revealed that changes in hydration status over 3 days affected urea transporter protein expression without changing its subcellular distribution.


Lim, S. W.; Han, K. H.; Jung, J. Y.; Kim, W. Y.; Yang, C. W.; Sands, J. M.; Knepper, M. A.; Madsen, K. M., and Kim, J.: Ultrastructural localization of UT-A and UT-B in rat kidneys with different hydration status. Am. J. Physiol. Regul. Integr. Comp. Physiol., 290, R479-492 (2006).

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