N A N O P R O B E S     E - N E W S

Vol. 7, No. 9          September 21, 2006

Microsporida are parasitic organisms that are important opportunistic pathogens in AIDS and other immune compromised patients. They are responsible for chronic diarrhea, malabsorption syndromes, myositis, and disseminating infections demonstrated in all tissues of the body. Approximately a dozen different microsporidia infect humans. All form a diagnostic spore containing a coiled polar filament surrounding the single nucleus or paired abutted nuclei (diplokaryon) and its associated cytoplasmic organelles, the sporoplasm; upon germination, the polar filament is everted to become a tubule, through which the spore contents travel to become the infective sporoplasm.

Cultured RK-13 cells infected with E. hellem microsporidia were grown on slides, immunofixed (for electron microscopy), and stored in PBS buffer. These were incubated with primary antibody (anti polar tube PTP-55 [6], 1:100) for one hour. A universal detection system incorporating a biotinylated secondary antibody and polymerized peroxidase-streptavidin detection (I-View, Ventana Medical Systems) was applied. After washing with PBS-0.01% Tween-20, distilled water, and 0.02 M sodium citrate buffer at pH 3.8, specimens were developed with a modified formulation of the enzyme metallographic reagent (Nanoprobes, Incorporated), washed again with 0.02 M sodium citrate buffer at pH 3.8, rinsed with deionized water, and coverslipped. After light microscope examination, areas of interest were marked on the back of the slides. The cover glasses were removed, and the slides rinsed in distilled water, dehydrated through a series of ethanol solutions (50% - 100%) and infiltrated with Araldite 502 resin (EMS, PA) overnight. Marked areas were covered with BEEM capsules filled with resin, and embedded at 60C for 24 hours. Thin sections were cut, placed on copper grids, and stained with uranyl acetate and lead citrate. Samples were examined using a transmission electron microscope. Some results are shown below.

The polar tubes were easily observed with brightfield optics, and background was very clean: almost no non-specific deposits were observed on the cells and surrounding matrix. The enzyme metallography visualization is more sensitive, with lower background and higher resolution than DAB staining. This method offers the promise of both a superior research tool for ultrastructural examination, and a simplified light microscopic assay for Microsporidian infection.

Reference:

• Powell, R.; Joshi, V.; Thelian, A.; Liu, W.; Takvorian, P.; Cali, A., and Hainfeld, J.: Light and Electron Microscopy of Microsporida using Enzyme Metallography. Microsc. Microanal., 12, (Suppl. 2: Proceedings); Kotula, P.; Marko, M.; Scott, J.-H.; Gauvin, R.; Beniac, D.; Lucas, G.; McKernan, S., and Shields, J. (Eds.), Cambridge University Press, New York, NY, 424CD (2006). (view as html)

More information:

• Complete paper from Microscopy & Microanalysis
• Enzyme metallography - paper from Microscopy & Microanalysis 2002
• Enzyme metallography - paper from Microscopy & Microanalysis 2004

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Polyfunctional Nanogold®

Although most users need monofunctional Nanogold® labeling reagents, a number of people have requested polyfunctional Nanogold - either the maleimido- or Sulfo-NHS derivatives. Although we do not offer these as catalog products, Nanoprobes already offers a polyfunctional Nanogold® reagent, Positively Charged Nanogold. This molecule contains a number of primary aliphatic amines: it is estimated that each Nanogold particle has up to 6 amines available for reaction. While it is primarily intended for staining negatively charged targets such as oligonucleotides, these amines can also be used for activation and cross-linking to biological molecules. Some of the reactions that are available are show below.

To conjugate biomolecules directly to Positively Charged Nanogold, the reaction used in peptide synthesis usually works well - react the carboxylic acid with EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide Hydrochloride) and Sulfo-NHS to convert it to a reactive Sulfo-N-hydroxysuccinimide ester. EDC is available from a number of suppliers, including Pierce, who provide a guide to its use. EDC reacts with a carboxyl group on the molecule to be labeled, forming an amine-reactive O-acylisourea intermediate. This intermediate could then react with Monoamino Nanogold; however, it is susceptible to hydrolysis, making it short-lived in aqueous solution. The addition of Sulfo-NHS (5 mM) stabilizes the amine-reactive intermediate by converting it to an amine-reactive Sulfo-NHS ester, increasing the efficiency of EDC-mediated coupling reactions.

Alternatively, the amino- groups on Positively Charged Nanogold may be activated using cross-linking reagents. Two examples are shown. In the first, the water-soluble homobifunctional cross-linking reagent bis (sulfo-succinimidyl) suberate (BS³) is used for conjugation to the N-terminal of a peptide in which the C-terminal has been protected. One feature of this procedure is that it provides a relatively long link: the C₈ chain of the BS³ is added to the propyl group which bears the amine; the propyl group projects beyond the outer surface of the ligand shell of the Nanogold particle. In the second scheme, a water-soluble heterobifunctional cross-linker, Sulfo-succinimidyl 4-[N-maleimido-methyl]-cyclohexane-1-carboxylate (Sulfo-SMCC), is used to convert the amines to maleimide groups, which have a highly specific reactivity towards thiols. The maleimido- derivative is then used to label a thiolated biomolecule such as a cysteine-containing peptide or a thiol-modified oligonucleotide.

Other cross-linking reactions are feasible, including labeling aldehydes and ketones or hydroxyls. You can find suitable cross-linkers from the list of heterobifunctional cross-linkers from Molecular Biosciences, or the cross-linker selection guide from Pierce.

If the molecule you wish to label contains an accessible aliphatic amine in a desirable position for labeling, you can also use Negatively Charged Nanogold to label it. Negatively Charged Nanogold contains multiple carboxylic acid groups; these may be activated by reaction with EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide Hydrochloride) and Sulfo-NHS to convert it to a reactive Sulfo-N-hydroxysuccinimide ester. You can purchase EDC from a number of sources; its use is described by Pierce. EDC reacts with a Nanogold carboxyl group, forming an amine-reactive O-acylisourea intermediate. This intermediate could then react with an amino-containing biomolecule; however, it is also susceptible to hydrolysis, making it unstable and short-lived in aqueous solution. The addition of Sulfo-NHS (5 mM) stabilizes this intermediate by converting it to an amine-reactive Sulfo-NHS ester, thus increasing the efficiency of EDC-mediated coupling reactions.

More information:

• Charged Nanogold: catalog and general information
• Positively Charged Nanogold: product and technical information
• Negatively Charged Nanogold: product and technical information
• Nanogold labeling reagents: technical help
• DNA Nanowires: M&M01 paper on the application of Positively Charged Nanogold
• Guide to Gold Cluster Labeling

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nanogold® and Lyosomal Storage Disease

There are 9 mammalian CLC proteins, which occur on the plasma membrane and intracellular vesicles. They function as Cl^- channels or as electrogenic Cl^-/H⁺ exchangers. ClC-6 is the least understood; its mRNA is found in many tissues, including brain and kidney. Upon heterologous expression, the ClC-6 protein is reported to colocalize with markers either of the endoplasmic reticulum or of endosomes. In their recent paper in Proceedings of the National Academy of Sciences of the USA, Poët and group use ClC-6 knockout (KO) mice to investigate the localization and function of this protein. The mice displayed a progressive neuropathy of the central and peripheral nervous systems, with features of neuronal ceroid lipofuscinosis (NCL), a subtype of human lysosomal storage disease, and presented a neurological phenotype consisting of impairment of pain sensation. In situ mRNA hybridization on whole mount sections showed intense labeling of the nervous system, including brain, trigeminal and dorsal root ganglia (DRG), spinal cord, and eye. Immunohistochemistry of adult mouse brain revealed that ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, particularly in dorsal root ganglia: a broad expression of the ClC-6 protein was found, including neurons in every layer in the cortex and all regions of the hippocampus. ClC-6 was found to colocalize with markers for late endosomes in neuronal cell bodies. Subcellular distribution of ClC-6 was independently analyzed by fractionating brain membranes using Percoll centrifugation, and it was concluded that ClC-6 is predominantly expressed in late endosomes. Golgi staining revealed frequent enlargements of the proximal axon of cortical neurons; electron microscopy identified lipopigment deposits as the likely cause. Similar "meganeurites" have been described in various lysosomal storage diseases, including NCL. Higher magnification showed a mixed composition, with lipid droplets associated with amorphous or granular material (granular osmiophilic deposits, or GRODs).

Immuonelectron microscopy was used to investigate the distribution of lyosomal markers associated with NCL. Lamp-1, cathepsin D, and ClC-7 were localized using Nanogold pre-embedding labeling with silver enhancement; Subunit C (subC) was localized with postembedding immuoelectron microscopy using protein A - colloidal gold. For pre-embedding immunoelectron microscopy, mice were perfused with a mixture of 4% paraformaldehyde and either 0.1% (for immunogold labeling) or 3% [for structural analysis of the brain and the dorsal root ganglia (DRG) in PBS] glutaraldehyde. 500 or 50 µm-thick sagittal vibratome sections were prepared from brain. For preembedding immunolabeling, the sections were cryoprotected in an ascending series of 0.5, 1, 1.5 and 2 M sucrose and subjected to two cycles of liquid nitrogen freeze-thaw to aid penetration of immunoreagents into the cells. Sections were blocked for 1 hour in 10% normal goat serum and 0.2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), then incubated with primary antibodies [cathepsin D (1:20), lamp1 (1:200), or the ClC-7 antibody 7N4B (1:200)] in carrier (PBS with 1% serum and 0,2% BSA) at 4°C overnight. Nanogold-labeled Goat anti-Rabbit antibody was then applied at 1:100 dilution for 2 hours; after washing, it was enhanced with the HQ Silver, followed by gold toning with 0.05% gold chloride in 150 mM Na-acetate buffer. After fixation with 1% osmium tetroxide, slides were dehydrated in an ascending series of ethanol and embedded in Epon. Ultrathin sections were then examined in the transmission electron microscope.

For postembedding immunogold, sections were prepared as described above up to sucrose infiltration, then freeze-slammed onto a polished copper plate precooled in liquid nitrogen and transferred into glass vials containing 0.5% uranyl acetate in methanol, precooled to -80°C and incubated for 1 hour. Thereafter, they were washed three times in methanol and precooled to -80°C. Sections were then embedded with Lowicryl HM20 and polymerized according to the procedure of Nusser. Specimens were immunolabeled with subunit C (subC) antibody (1:20) and 10 nm large protein A gold on ultrathin sections collected on copper grids coated with pioloform. LAP activity in situ was determined using beta-glycerophosphate as a substrate. Tissues were incubated for 2 hours at 37°C in 8.5 mM sodium-beta-glycerophosphate with 3 mM silver nitrate in 50 mM acetate buffer pH 5.0. After washing with water followed by 1% Na₂S, then 50 mM sodium acetate and PBS, samples were incubated with 1% osmium tetroxide for 20 min, dehydrated, and embedded in Epon resin. No staining was observed when the substrate was omitted.

These studies indicated the presence of lysosomal proteins and other marker proteins typical for NCL in the electron-dense storage material that caused the pathological enlargement of proximal axons. This indicates that CLCN6 is a candidate gene for mild forms of human NCL. An analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients, but failed to prove a causative role of CLCN6 in that disease.

Reference:

• Poët, M.; Kornak, U.; Schweizer, M.; Zdebik, A. A.; Scheel, O.; Hoelter, S.; Wurst, W.; Schmitt, A.; Fuhrmann, J. C.; Planells-Cases, R.; Mole, S. E.; Hübner, C. A., and Jentsch, T. J.: Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. Proc. Natl. Acad. Sci. USA,/cite> 103, 13854-13859 (2006).
  • Abstract (courtesy of Proceedings of the National Adacemy of Sciences of the USA

More information:

• Nanogold conjugates: Catalog and general information
• Nanogold-antibody conjugates: product and technical information
• Silver enhancers: Catalog and general information
• HQ Silver: product and technical information
• Technical help: Nanogold conjugates
• Nanogold labeling references, sorted by application

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nanogold® and Undecagold Beams for Matter Wave Interferometry

Reiger and co-workers have investigated Nanogold® and Undecagold using three well-established molecular beam methods, and describe their findings in a recent issue of Optics Communications. The authors studied beams of gold nanoclusters generated by electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI) and direct thermal laser desorption (TLD). These source methods are complemented by three different detection techniques, respectively quadrupole mass spectroscopy (QMS), time-of-flight mass spectroscopy (TOF-MS) and multi-photon ionization (MUPI) TOFMS. Undecagold and Nanogold were selected for study because they are thought to be particularly stable as they are ligand-stabilized, and were used without any further purification.

ESI was conducted using a source operated with needles with a diameter of 30 µm, and flow rates below 1 µl/min. Samples of Non-functional Nanogold or Non-functional Undecagold were dissolved in methanol with typical gold crystal concentrations of c = 1 - 5 x 10^-5mol/l. The unsolvated gold nanocrystals were then electrostatically guided by a custom made air/vacuum interface, through two differentially pumped vacuum stages into a high vacuum region, where they could be selected and detected in a quadrupole mass spectrometer. In a typical mass spectrum of undecagold, the expected peak of [Au₁₁(PAr₃)₇Cl₃] (5307 amu) was observed, as well as one well-defined peak consistent with an ionic composition [Au₁₁(PAr₃)₈Cl₂]Cl (5740 amu) and some [Au₁₃(PAr₃)₇Cl₄] (5736 amu). In a larger mass scan, all charge states from 1+ through 4+ could be observed, but with negligible formation of fragments or major aggregates. The flux through the quadrupole, integrated over the four charge states, amounts to more than one million mass selected and detected gold particles per second, sufficiently high to load an ion trap in a few seconds. The larger Nanogold crystals were also well volatilized by electrospray. Three charge groups, corresponding to 2+ through 4+, were identified: each charge group was composed of up to 20 different peaks, with a periodic spacing between adjacent peaks consistent with the addition or subtraction of single gold atoms and/or single ligands. Peak cluster compositions ranged from [Au₅₅L12Cl6] to [Au₄₄L₈Cl₆].

Matrix assisted laser desorption ionization, MALDI, conducted using ultraviolet laser light irradiated on a strongly absorbing organic matrix, heats the sample in a few nanoseconds to give abrupt evaporation. Samples were prepared using the dried droplet method and dihydroxy benzoic acid (DHB), a common MALDI matrix. The sample plate was transferred into a high vacuum desorption chamber and irradiated by a pulsed N₂-laser beam (k = 337 nm, 4 ns, 5 Hz, 250 µm) with an energy of 3 µJ. At lower desorption energy, no significant ion signal was observed. The emerging particles were extracted into an orthogonal time-of-flight mass spectrometer. The ion mass spectrum for undecagold contained a peak attributed to the singly charged intact cluster, and 10 fragment peaks attributed to the loss of PAr3 ligands and the successive losses of gold-chlorine complexes. Combination peaks of nanocrystal fragments were seen at high masses. Similar results were obtained with Nanogold, indicating that, although UV-MALDI is a well-established method for protein characterization, it is more invasive for gold clusters. This was thought to be due to the plasmon resonance in gold, which gives strong absorption at the desorption wavelength.

MALDI produces a significant fraction of neutral particles, which could be used for quantum interference experiments, but the small analyte concentration and large beam divergence make their detection difficult. However, by omitting the matrix, the gold concentration in the beam can be increased up to 10,000-fold. For this method, thermal laser desorption (TLD), the methanol-gold solution is directly dried on the sample plate: the methanol almost completely evaporates, leaving behind a pure layer of ligand-stabilized gold clusters. With the same laser and procedure used previously for MALDI, TLD resulted in the formation of significant amounts of neutral and ligand-free gold clusters ranging from [Au₂] to [Au₂₅]. Highest cluster yield was obtained for a desorption laser intensity around 10 µJ focused to 200 µm. Neutrals were detected by postionizing them with a pulsed Nd-YAG laser (266 nm, 68 ns, 1 - 4 mJ) focused to a spot of about 1 mm diameter at the entrance region of the TOF-MS, located 30 mm behind the desorption region. Up to a common scaling factor of 2.4, identical spectra were obtained for undecagold and Nanogold: no significant signal was observed beyond 5000 u, and no signature of clusters with an intact ligand shell. The velocity distribution of the neutral beam was measured by observing the signal strength for varying delay times between the desorption and the ionization laser pulse: typical mean velocities were deduced to be from v_mp ~ 550 m/s for the smallest clusters [Au₂] to v_mp ~ 450 m/s for [Au₁₃].

Matter wave physics is experimentally facilitated by using neutral, slow and mass selected particles. Interferometry experiments with electrons and He⁺ ions show that it may also be conceivable with charged Nanogold; however, the use of neutral particles eliminates the need of shielding against electro-magnetic perturbations, and the authors consider the prospects of the three beam methods for matter wave interferometry. Direct thermal laser desorption generates a beam of a large range of ligand-free neutral gold clusters, with a signal strength sufficient for near-field interferometry with small clusters, and is the most promising method. Matrix assisted laser desorption also allows the volatilization of ligand-stabilized gold, but does not generate pure or monodisperse particles. Electrospray ionization is a minimally invasive technique for volatilizing gold nanoparticles, and permits the preparation of beams of size-selected large, charged nanocrystals; combined with ligand removal by oxygen plasma, this provides a feasible method for generating suitable beams. The authors provide a detailed proposal, using modifications of these methods and instruments, for an experimental system that may be used for particles in the mass range of 10⁶ amu.

Reference:

• Reiger, E.; Hackermüller, L.; Berninger, M.; and Arndt, M.: Exploration of gold nanoparticle beams for matter wave interferometry. Optics Communications, 264, 326-332 (2006).
  • Abstract (courtesy of Science Direct).

More information:

• Nanogold labeling reagents: catalog information
• Undecagold: catalog information
• Non-functional Nanogold: product information and instructions
• Non-functional Undecagold: product information and instructions
• Technical help: Nanogold labeling reagents
• Technical help: Undecagold

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Other Recent Publications

Veit, Keene and co-workers used GoldEnhance to enlarge small colloidal gold particles used as a marker for collagens in their studies on the interaction sites of Collagen XII, a multidomain protein with a small collagenous region known to interact with fibrillar collagens through its C-terminal region. No interactions to other extracellular proteins have been identified involving the non-collagenous N-terminal NC3 domain; to further elucidate the components of protein complexes that occur close to collagen fibrils, a number of different extracellular matrix proteins were tested for interaction in a solid phase assay. Binding to the NC3 domain of collagen XII was found for the avian homologue of tenascin-X; in humans, this is linked to Ehlers-Danlos disease. Binding was further characterized by surface plasmon resonance spectroscopy and supported by immunohistochemical co-localization in chick and mouse tissue. Pre-embedding 1 nm colloidal gold labeling with gold enhancement and with 10 nm colloidal gold was then used to detect and localize collagen XII and tenascin-X: Collagen XII in both chick dermis and in mouse dermis is located either directly on banded fibrils or integrated in electron-dense material that is attached to the fibrils, and Tenascin-X in chick dermis and in mouse dermis shows a similar localization. This confirms the novel interaction, and also shows that GoldEnhance is effective for enhancement of other small colloidal gold in addition to Nanogold.

Reference:

• Veit, G.; Hansen, U.; Keene, D. R.; Bruckner, P.; Chiquet-Ehrismann, R.; Chiquet, M., and Koch, M.: Collagen XII Interacts with Avian Tenascin-X through Its NC3 Domain. J. Biol. Chem., 281, 27461-27470 (2006).
  • Abstract (courtesy of the Journal of Biological Chemistry).

We are always interested in the exploits of Gorm Danscher in the development of autometallography (AMG), and his latest report in Progress in Histochemistry and Cytochemistry, in which he uses autometallographic methods to visualize quantum dots, does not disappoint. Autometallographic (AMG) silver enhancement is a potent histochemical tool which he has refined for tracing a variety of metal containing nanocrystals, including pure gold and silver nanoclusters, and quantum dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium. Such nanocrystals can be created in many different ways; they include manufacturing colloidal gold or silver particles, treating an organism in vivo with sulfide or selenide ions, as the product of metabolic decomposition of bismuth-, mercury- or silver-containing macromolecules in cell organelles, or as the end product of histochemical processing of tissue sections. This article provides a comprehensive review of the autometallographic silver amplification techniques known today, and their use in biology, from the introduction of Timms "silver-sulphide staining" in 1958, through its evolution and expansion into several different areas of research. Applications in immunocytochemistry, tracing of enzymes at LM and EM levels, blot staining, retrograde axonal tracing of zinc-enriched (ZEN) neurons, counterstaining of semithin sections, enhancement of histochemical reaction products, marking of phagocytotic cells, staining of myelin, tracing of gold ions released from gold implants, and visualization of capillaries are discussed. Technical comments and protocols for current methods, with a summary of the most significant scientific results obtained by this range of AMG histochemical approaches are included.

Reference:

• Danscher, G., and Stoltenberg, M.: Silver enhancement of quantum dots resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, in vitro and immersion created zinc-sulphur/zinc-selenium nanocrystals, (3) metal ions liberated from metal implants and particles. Prog. Histochem. Cytochem., 41, 57-139 (2006).
  • Abstract (courtesy of Science Direct).

Meanwhile, in Biosensors and Bioelectronics, Cao and co-workers report a new biochip assay using silver-enhanced colloidal gold, read either visually or using an optical scanner. DNA microarrays, coupled with multiplex asymmetrical PCR, were developed for the simultaneous, sensitive and specific detection of Ureaplasma urealyticum and Chlamydia trachomatis. 5'-amino-modified oligonucleotides, immobilized on glass surface, acted as capturing probes: these were designed to bind complementary, biotinylated target DNA which was then detected using streptavidin labeled with colloidal gold 8 to 15 nm in diameter. These were then washed with 2PBN (0.3M NaNO₃, 10mM NaH₂PO₄/Na₂HPO4 buffer, pH 7.0) and three times (2 min each time) with double-distilled water, then incubated for 10-15 minutes at room temperature in silver blue solution, a colorimetric solution, containing maleate buffer, silver salt (AgNO₃) and hydroquinone, was prepared as described previously. The slides were rinsed in water, air-dried for 5 hours at 37°C, and read with a visible light scanner or observed visually. Multiplex asymmetrical PCR of U. urealyticum, C. trachomatis and Bacillus subtilis (positive control) was used to prepare biotinylated single-stranded target DNA. Several clinical samples of U. urealyticum and C. trachomatis from infected patients were tested using home-made DNA microarrays. The assay combined high sensitivity, detecting as little as 5 pM concentrations of target DNA, with good specificity, simplicity, cheapness and speed, giving it potential applications in the clinical field.

Reference:

• Cao, X.; Wang, Y. F.; Zhang, C. F., and Gao, W. J.: Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR. Biosens. Bioelectron., 22, 393-398 (2006).
  • Abstract (courtesy of Science Direct).

Reference for silver blue preparation:

• Wang, Y. F.; Pang, D. W.; Zhang, Z. L.; Zheng, H. Z.; Cao, J. P., and Shen, J. T.: Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement. J. Med. Virol., 70, 205211 (2003).
  • Abstract (courtesy of Wiley Interscience).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .