N A N O P R O B E S E - N E W S
Vol. 8, No. 1 January 19, 2007
This probe has several significant advantages over conventional antibody probes:
- Higher labeling resolution. The nitrilotriacetic acid - Ni(II) chelate is much smaller than an antibody or protein, and therefore when it is bound, the gold is much closer to its target. This makes NTA-Ni(II)-Nanogold ideal for localizing sites in protein complexes or other macromolecular assemblies at molecular resolution.
- Better penetration: because it is so small, NTA-Ni(II)-Nanogold can more easily penetrate into specimens and access sterically restricted sites within specimens, and perturbs their ultrastructure less. In some systems it may be used with stronger fixation or less permeabilization, enabling labeling with better ultrastructural preservation.
- NTA-Ni(II)-Nanogold is prepared using a modified gold particle, with very high solubility and stability. At 1.8 nm in size, it is readily visualized by electron microscopy.
- Binding constants for Ni(II)-NTA are very high due to the combination of the chelate effect of multiple histidine binding, and target binding of 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.
In the first paper, in PNAS, Valerie Pye and co-workers in the used this probe to study the interactions of ATPases. p97/VCP (Cdc48 in yeast) is an essential and abundant AAA+ ATPase which is involved in a several cellular pathways through interactions with different adaptor proteins. The two best characterized adaptors are p47, which directs p97 to membrane fusion events and has been shown to be involved in protein degradation, and the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization 4) complex, which directs p97 to an essential role in endoplasmic reticulum-associated degradation (ERAD), and an important role in postmitotic spindle disassembly. While p47 has been relatively well characterized, little is known about the structure of the Ufd1-Npl4 complex and its interaction with p97. The authors used mass spectrometry, NTA-Ni(II)-Nanogold labeling with cryoelectron microscopy, single-particle negative stain transmission electron microscopy (TEM) with antibody labeling, and scanning transmission electron microscopy (STEM) to produce the first low-resolution structure of the Ufd1-Npl4 complex and how it interacts with p97.
Characterizing Ufd1-Npl4 using electrospray ionization MS revealed a major species with mass of 103.1 kDa and two minor species of 34.8 and 68.1 kDa. These masses correspond to the Ufd1-Npl4 complex, the Ufd1 monomer, and the Npl4 monomer respectively. No peaks corresponding to a 2:2 or 3:3 complex were observed, establishing a 1:1 ratio for the complex. Examination by cryoelectron microscopy with uranyl acetate negative staining showed a homogeneous population of Ufd1-Npl4 heterodimers possessing an elongated bilobed structure, ~80 ± 30 Å in length. Confirmation that these particles were Ufd1 was achieved using Ufd1(His6) expressed in Escherichia coli Rosetta (DE3) cells, purified by separation of clarified cell lysates over a NTA chelating column precharged with Ni2+ eluted with an imidazole gradient. Purified Ufd1(His6)-Npl4, diluted to ~ 20 µg/ml in 150 mM KCl, 25 mM Tris, 2.5 mM MgCl2, and 1 mM ATP (pH 8.0) were adsorbed onto a glow-discharged carbon-coated grids and incubated for 10 minutes with sub-stoichiometric amounts of NTA-Ni(II)-Nanogold. The grids were then washed quickly with buffer followed by two drops of water, negatively stained with 1% uranyl acetate, blotted, and air dried. Drops (5 µl) of Ufd1-Npl4 sample were applied to washed glow-discharged copper grids supporting a lacey carbon film, blotted quickly with filter paper and quench-frozen in liquid ethane. The grids were transferred to a cryo-holder; micrographs were recorded at an accelerating voltage of 200 kV at a nominal magnification of 50,000 X on film under minimal electron dose illumination, then digitized to 2.6 Å per pixel. One Nanogold particle was observed per particle superimposed with one lobe of density.
left: Structure of Ni-NTA-Nanogold, showing interaction with a His-tagged protein. Inset shows structure of p97-Ufd1-Npl4 labeled with NTA-Ni(II)-Nanogold, showing resolution. right: 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).
The authors confirmed their identification by using a monoclonal antibody directed against the region between the subdomains of Ufd1 (amino acids 211220), and observing binding by negative stain TEM. The antibody was found to recognize an epitope located in the middle of the Ufd1-Npl4: in the negative stain images, the antibody was clearly visualized binding to a site in the middle of the elongated particle. The mass of the Ufd1 particles was also confirmed using scanning transmission electron microscopy, which allows accurate mass measurements: given absolute scattering cross-sections, intensity can be integrated over an isolated particle and converted to a molecular weight. Analysis of 800 particles using the PCMass software confirmed the MW of about 100 kDa.
Using negative stain TEM, one Ufd1-Npl4 heterodimer was shown to interact with one p97 hexamer to form the p97-Ufd1-Npl4 complex: the Ufd1-Npl4 heterodimer was clearly visualized to emanate from one region on the periphery of the N-D1 plane of the p97 hexamer. Binding to P97 was then confirmed by labeling the p97-Ufd1-Npl4 complex with NTA-Ni(II)-Nanogold to target the C-terminal His6-tag of Ufd1: a single NTA-Ni(II)-Nanogold particle was observed at the periphery of each p97 hexamer. A purified IgG targeting the C-terminal zinc finger of Npl4, and a monoclonal antibody targeting Ufd1 were also observed by negative stain TEM to bind the complex. Both mono- and poly-ubiquitin binding were observed at a similar location within Ufd1-Npl4 complex, consistent with earlier findings that the complex posseses two different ubiquitin binding sites. The p97-p47 and p97-Ufd1-Npl4 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting differences in their specific actions and their ability to interact with additional cofactors that cooperate with p97 in different cellular pathways.
- Pye, V. E, Beuron, F, Keetch, C. A, McKeown, C, Robinson, C. V, Meyer, H. H, Zhang, X, and Freemont, P. S.: Structural insights into the p97-Ufd1-Npl4 complex. Proc. Natl. Acad. Sci. USA, 104, 467-472 (2007).
Meanwhile Frye, Collins and co-workers followed up on their earlier studies on the Role of Wzc in the Assembly of e.coli Group 1 Capsules with a study on the topology of the outer-membrane secretin PilQ from Neisseria meningitidis, the causative agent of epidemic meningococcal meningitis and septicemia. Type IV pili are surface organelles that mediate a variety of functions, including adhesion, twitching motility, and competence for DNA binding and uptake in transformation. The secretin PilQ is required for type IV pilus expression at the cell surface: it forms a dodecameric cage-like macromolecular complex in the meningococcal outer membrane. PilQ-null mutants have no surface pili, and it is thought that the PilQ complex facilitates extrusion and retraction of type IV pili across the outer membrane.
The cellular localization was confirmed by immunofluorescence, and electron microscopy labeling with colloidal gold using the antibody K010, which recognizes N-terminal PilQ, showed that this domain of PilQ was localized in the inner leaf of the outer membrane, facing the periplasm. The PilQ protein could also be visualized associated with the extracellular face of the inner membrane, and these results strongly imply that N-terminal PilQ is situated in the periplasm of the meningococcal cell.
In order to understand the structurefunction relationships of this important protein in pilus biology, it is necessary to know the orientation of the meningococcal PilQ complex in the membrane. The authors take the first step to defining the topology of the PilQ complex in the outer membrane by using polyhistidine insertions in N- and C-terminal regions of PilQ and examining their subcellular locations using NTA-Ni(II)-Nanogold, visualized by cryoelectron microscopy; this achieves the greater resolution required for this work. His-tag insertions were designed to insert into the predicted random coils of the expressed protein. For the generation of internally His-tagged PilQ splicing by overlapping extension was used. The 39 and 59 fragments were produced with the primer combinations SF9/SF16 and SF17/SF12b, PilQ1/PilQ2 and PilQ3/PilQ4c, and PilQ1/SF4 and SF5/PilQ4c, for constructs Mc-205, Mc-656 and Mc-678, respectively. The SOEing PCR final products were amplified with the external primers SF9/SF12b, PilQ1/PilQ4c and PilQ1/PilQ4c for constructs Mc-205, Mc-656 and Mc-678, respectively, and cloned into the vector pUP6. Final constructs were used for transformation of WT Mc-M1080. NTA-Ni(II)-Nanogold labeling of PilQ was performed by incubating a 10 µL volume of His6-PilQ (100 µg mL) from Mc-205 with 5 µL of NTA-Ni(II)-Nanogold for 24 hours at 4°C, with agitation. Samples were then centrifuged at 16,000 g for 5 minutes, then prepared for cryo-negative staining, as previously described (Collins et al., 2006). Grids were placed in a cryo-stage, and data recorded at <100 K in a FEG CM200 microscope, used in conjunction with a 4K CCD camera.
Individual gold-labelled particles were interactively selected in 64 pixel boxes (Å per pixel, 3.1) using BOXER. Following contrast transfer function (CTF) correction, data were contrast normalized, centred, and low-pass filtered to 25 Å resolution. After applying a 190 Å circular mask, six rounds of iterative refinement were performed in C4 symmetry, using the previously calculated 3D structure of PilQ filtered to 25 Å resolution as a start model.
Notably, the insertion epitopes at residues 205 and 678 were located within the periplasm, whereas residue 656 was exposed at the outer surface of the outer membrane. NTA-Ni(II)-Nanogold demonstrated that the insertion at residue 205 within the N-terminus mapped to a site on the arm-like features of the 3D structure of the PilQ multimer. Interestingly, mutation of the same region gave rise to an increase in vancomycin permeability through the PilQ complex. The results yield novel information on the PilQ N-terminal location and function in the periplasm, and reveal a complex organization of the membrane-spanning secretin in vivo.
- Frye, S. A.; Assalkhou, R, Collins, R. F.; Ford, R. C.; Petersson, C, Derrick, J. P.; Tonjum, T.: Topology of the outer-membrane secretin PilQ from Neisseria meningitidis.
Microbiology. 2006 Dec;152(Pt 12):3751-64. (2007).
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The target binding interaction of NTA-Ni(II)-Nanogold is based on the coordination of ligands to the central nickel (II) ion through electronegative groups or groups containing electron lone pairs, particularly aromatic nitrogens. This differs significantly from the binding mechanism of antibodies, which usually functions through protein-protein hydrogen-bonding based interactions, or other targeted biomolecules such as streptavidin. Therefore binding is moderated or reversed by different reagents and conditions, and the most effective blocking and washing reagents for this product are different to those used for antibodies and proteins. Furthermore, the Binding occurs by coordination of electronegative atoms, usually aromatic nitrogen, to the nickel (II) ion; if you are labeling a target that contains other aromatic nitrogens, such as other histidine residues, these may also bind. The undesirable interaction may be eliminated by treatments that reduce this interaction. The following steps may be useful:
If you are seeing background signal after silver or gold enhancement, a number of methods are available for stopping these reactions and preventing further reaction after the desired end-point by reagents that have diffused into specimens.
- Wash with a buffer containing imidazole. Imidazole is the active coordinating group in histidine; treatment with imidazole will displace isolated single histidine binding, but it will not overcome the chelate effect and stronger binding of NTA with polyhistidines, and therefore will disturb polyhistidine binding much less. Try increasing the imidazole concentration from 10 mM progressively to 200 mM until background is controlled to your satisfaction.
- Increase the ionic strength of the solution. The nitrilotriacetic acid moiety is negatively charged, and may interact with positively charged regions of a target; increasing the ionic strength will help to prevent this. Try 300 mM NaCl, and, if your biomolecule can tolerate it, increase to 1.0 M if necessary.
- Other possible factors include:
- hydrophobic interactions. These may be reduced or eliminated by the addition of detergents; try 0.05% Tween-20, and increase to 0.1% if necessary.
- pH: it may be helpful to vary the pH to find an optimum pH at which charge interactions such as those mentioned above are reduced.
- Transition metals may also promote interactions between the NTA group and electron donating groups in your specimen. Wash with 0.05 M disodium ethylene diamine tetraacetic acid (EDTA) to remove these.
- Interaction of thiols (sulfhydryls) with gold. Thiols have a strong affinity for gold, and if they are present in your specimen, any gold particle species, including Ni-NTA-Nanogold, may bind to them. Avoid the use of reducing buffers or preservatives containing thiols, such as dithiothreitol (DTT), mercaptoethanol, or mercaptoethylamine hydrochloride. If your specimen contains exposed thiols, they may be blocked with N-ethylmaleimide.
In the development of Ni-NTA-Nanogold, it was found that a form containing multiple NTA-Ni(II) groups produced the best overall combination of labeling selectivity, density and sensitivity. However, because this can interact with polyhistidine tags on several protein molecules simultaneously, it may act to aggregate proteins, or perturb the formation of protein complexes, in solution. The best approach to avoid this is to use a ratio of reagent to protein such that the stoichiometry reduces or eliminates this possibility. For example, if your protein has only one polyhistidine tag, then using an excess of the Ni-NTA-Nanogold reagent will guard against the possibility of multiple interactions. You can also help avoid the possibility by carefully selecting when to add the reagent, for example after complex assembly.
- Hainfeld, J. F.; Liu, W.; Halsey, C. M. R.; Freimuth, P., and Powell, R. D.: Ni-NTA-Gold Clusters Target His-Tagged Proteins. J. Struct. Biol., 127, 185-198 (1999).
- Buchel, C.; Morris, E.; Orlova, E.; and Barber, J.: Localisation of the PsbH subunit in photosystem II: a new approach using labelling of His-tags with a Ni(2+)-NTA gold cluster and single particle analysis. J. Mol. Biol., 312, 371-379 (2001).
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
If FluoroNanogold is not suited for your experiment but you need to correlate fluorescence with electron microscopic localization, an alternative is to use a Green Fluorescent Protein (GFP) fusion protein to localize your target by fluorescence, then localize this using a Nanogold conjugate targeted to the green fluorescent protein. Baldassarre and group report an application of this method in their recent paper on actin dynamics at sites of extracellular matrix degradation in the European Journal of Cell Biology.
Degradation of extracellular matrix (ECM) by proteases is an essential part of physiological and pathological cell invasion. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions, termed invadopodia. The authors present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation, and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia demonstrated the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover.
Correlative light-electron microscopy (CLEM) was performed as described in the earlier references given below. A375MM cells stably expressing the human beta-actin-GFP chimera were grown on MatTek glass bottom Petri dishes coated with fluorescent gelatin. After appropriate times, for in vivo experiments, samples were analyzed using a laser scanning confocal microscope and the cell bearing the structure of interest was localized within the grid coordinates. The cells were then fixed with 0.05% glutaraldehyde with 4% paraformaldehyde in 0.2M HEPES (pH 7.4) for 5 minutes, followed by 4% paraformaldehyde in the same buffer for 30 minutes. The cells were then incubated in 0.02% saponin-containing blocking solution (phosphate buffered saline (PBS) supplemented with 0.2% BSA and 50 mM NH4Cl); after washing, these samples were then incubated with anti-GFP antibodies, extensively washed, and revealed using Nanogold-labeled secondary antibodies followed by gold enhancement with GoldEnhance EM. Samples were then processed for conventional electron microscopy with 1% OsO4 plus 1.5% potassium ferrocyanide in 0.1M cacodylate buffer (pH 7.3) for 2 hours on ice in the dark, then dehydrated, embedded in epoxy resin and polymerized for at least 24 hours. Coverslips were then dissolved with 40% hydrofluoric acid and samples were extensively washed with buffer. Serial sections of the cell of interest were produced parallel to the substrate: 100 nm serial sections were collected on slot grids covered with Formvar-carbon supporting film, and examined in the electron microscope at 200 kV; images were collected with a slow-scan CCD camera. Images collected by confocal and electron microscopy were aligned with Adobe Photoshop and the structure of interest was identified on the basis of its position on the grid coordinates.
The results offer new insight into the tight coordination between signaling, actin remodeling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. Invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that are also experimentally valuable for the study of these processes.
- Baldassarre, M.; Ayala, I.; Beznoussenko, G.; Giacchetti, G.; Machesky, L. M.; Luini, A., and Buccione, R.: Actin dynamics at sites of extracellular matrix degradation. Eur. J. Cell. Biol., 85, 1217-1231 (2006).
- Polishchuk, R. S.; Polishchuk, E. V.; Marra, P.; Alberti, S.; Buccione, R.; Luini, A., and Mironov, A. A.: Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane. J. Cell Biol., 148, 45-58 (2000).
- Polishchuk, R. S., and Mironov, A. A.: Correlative Video Light/Electron Microscopy. Current Protocols in Cell Biology. In: Bonifacino, J. S.; Dasso, M.; Harford, J. B.; Lippincott-Schwartz, J., and Yamada, K. M. (Eds.), Current Protocols in Cell Biology, Wiley, New York, pp. 220.127.116.11.9 (2001).
* Alexa Fluor is a registered trademark of Invitrogen (Molecular Probes), Inc.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Electron tomography of immunolabeled proteins identified using amplified (enhanced) Nanogold® particles imaged by Scanning and Transmission Electron Microscopy within thick sections is a powerful method to investigate the three-dimensional organization of complex cellular structures. Tchelidze and colleagues, in their recent paper in the Journal of Structural Biology, describe two methods that improve the tomographic reconstruction process and hence increase the overall quality of the reconstructed cube. First, a very precise alignment of the projections was performed before reconstruction with a technique using sinograms. After reconstruction, a method is proposed to compute image restoration by calculating the Point Spread Function of the projection/back-projection system, and to use it to deblur the reconstructed cubes.
Electron tomography of complex molecular machineries within organelles requires image sections of several hundred to several thousand nanometers in thickness. Direct identification of molecules based on their shape is impossible in such specimens, due to the limited resolution of the tomograms and to the extremely high number of constitutive molecules present in the section (molecular crowding). These limitations impose a specific requirement for the identification of the molecules with a cytochemical or immunocytochemical labeling method using electron dense markers. Different types of electron dense end product are suited to different targets, depending on the size and the atomic number of the reaction end-product. For example, DAB/osmium precipitates allow investigation of the global shape of Golgi stacks containing enzymes identified through immunolabeling or through GFP-induced photooxidation; in earlier work, the authors demonstrated that Nucleolar Organizer Regions (NORs), identified by 5 - 10 nm silver dots (the end-product of the Ag-NOR cytochemical staining) could be imaged in sections several micrometers thick by using a 300 kV STEM, enabling investigation of the 3D organization of metaphasic NORs by electron tomography and a proposed model of their fine structure.
The high spatial resolution possible with Nanogold with silver enhancement enables further improvements in this process. Electron tomography based on pre-embedding labeling has many potential applications in the field of 3D investigation of GFP-tagged proteins identified with antibodies raised against GFP, as mentioned above: potentially, this can be used to investigate the 3D distribution of numerous nuclear machineries containing GFP-tagged proteins. However, before such an approach can be widely applied, it is necessary to increase the resolution of the tomograms obtained from amplified Nanogold particles imaged by STEM, and by providing the require improvement, these new methods enable these applications.
Improvement in the quality of the reconstructed cubes is demonstrated on images of nucleolar proteins tagged with EGFP and immunolabelled with nanogold particles. Full-length clones of hUBF1 were PCR-amplified. After a BspEIBamHI digest, the PCR products were cloned into the AgeI and BamHI sites of pEGFP-C1 vector (Clontech); the selected clone for UBF1 was sequenced on both strands. KB cells were grown without antibiotics in DMEM containing a source of L-glutamine and 10% fetal bovine serum (Invitrogen). Cells were seeded at 5104 cells/cm2 on glass coverslips, and incubated at 37°C in an atmosphere enriched with 5% CO2 for 2436 hours. At 5060% confluency, 1 µg plasmid DNA was introduced into the cells by transfection with FuGene-6 (Roche Diagnostics). After incubation for 24 hours at 37°C, cells were checked for dotted fluorescence in the fibrillar centers of the nucleoli; 50 ng/mL of actinomycin D, an inhibitor of RNA polymerase 1, was then added within the cell culture to induce segregation of nucleolar components.
Cells were fixed using a method which induces no redistribution of proteins as proved by comparing the localization of GFP-tagged proteins within the same cell before and after fixation: cells were fixed for 10 minutes in 4% paraformaldehyde diluted in phosphate-buffered saline (PBS), then permeabilized with 0.5% Triton X-100 in PBS for 5 minutes without light, soaked for 30 minutes in 3% BSA in PBS, then incubated for 30 minutes with anti-GFP mouse antibody diluted 1:50. Cells were then rinsed three times in PBS over 5 minutes. A biotinylated affinipure F(ab')2 fragment of goat anti-mouse IgG was applied at 1 : 100 for 30 minutes and visualized using a streptavidin-Texas-Red conjugate on one part of the cell culture to verify that GFP-tagged UBF1 was correctly immunolabeled; comparison of green (GFP-tagged UBF1) and red fluorescence (anti-GFP) confirmed the high efficiency of immunolabeling. On the other part of the cell culture, goat anti-mouse IgG was labeled with a Nanogold-Streptavidin diluted 1:500 in PBS. After fixation with 1.6% glutaraldehyde in PBS, cells were washed in deionized water and silver enhanced with HQ Silver to obtain 10 nm particles. Cells were collected by scraping, dehydrated in graded alcohols, embedded in Epikote 812, polymerised for 23 days at 60°C. Before performing electron tomography, 80 nm thick ultrathin sections were counterstained with lead citrate and uranyl acetate, then examined at 75 kV in the electron microscope to check for the preservation of cells and the precise localization of the goldsilver particles.
For electron tomography, sections, 500nm in thickness, were prepared and mounted on London finder copper grids. These sections were not counterstained with lead citrate and uranyl acetate, so only gold particles were imaged. These were then observed using a 250kV CM30 electron microscope working in the STEM mode. Sections were imaged at a direct 50,000 X magnification with a 5.6 nm electron beam. Before initiating a tilt series, the specimen was stabilized under the electron beam at a dose of 100 e-/(Å2xs) for 10 minutes to limit its anisotropic thinning during data collection. After correction of alignment of the eucentric goniometer stage, each section was tilted every 2° from -60° to +60°. Images were directly recorded on an onaxis high angular annular dark-field detector (HAADF) and digitized on line using Orion software (ORION Digital Image Acquisition System, SPI, Belgium). After focus and contrast control with high speed scanning, images (512 X 512 pixels) were collected using low speed scanning.
Reference for specimen preparation:
- Tchelidze, P.; Sauvage, C.; Bonnet, N.; Kilian, L.; Beorchia, A.; O'Donohue, MF.; Ploton, D., and Kaplan, H.: Electron tomography of amplified nanogold immunolabelling: Improvement of quality based on alignment of projections with sinograms and use of post-reconstruction deconvolution. J. Struct. Biol., 156, 421-431. (2006).
- Cheutin, T.; ODonohue, M. F.; Beorchia, A.; Vandelaer, M.; Kaplan, H.; Defever, B.; Ploton, D., and Thiry, M.: Three-dimensional organization of active rDNA genes within the nucleolus. J. Cell Sci., 115, 32973307 (2002).
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nanoprobes, Incorporated has received a new Phase 2 Small Business Innovation Research (SBIR) grant from the National Cancer Institute (National Institutes of Health) to develop gold nanoparticle-based imaging agents for Micro-Computed Tomography (MicroCT). The award of about $850,000 for two years will support the development of gold nanoparticle-based contrast agent products that will then be available to other researchers using microCT that will enable vascular and targeted imaging. A major goal is to make these agents available for microCT and eventual human use. If successful, this effort will produce superior blood pool agents and the first targeted CT contrast agent. The research will be conducted in collaboration with Henry Smilowitz, Ph.D. at the University Of Connecticut Health Center, Avraham Dilmanian, Ph.D. at Brookhaven National Laboratory, and Daniel Slatkin, MD.
Physically, gold is a better contrast element than the conventionally used iodine for both micro-CT and clinical CT. For micro-CT, the beam's energy spectrum can be tailored to be just above golds 12-14-keV L-edge (e.g. 40 kVp), while for clinical CT it can be tuned to be above gold's 80.7-keV K-edge (e.g., 150 kVp with Cu filtration), thus optimizing the image contrast-to-noise ratio for both applications. Using this approach, 20 m blood vessels in live animals have already been successfully imaged with microCT, and vascular casts obtained, a goal that has never before been achieved. Small orthotopic colon tumors were also detected in vivo using gold nanoparticles and microCT. The phamacokinetics and toxicity of the compounds have been found to be acceptable for human use development.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nano-W continues to become more widely used, and this month played a small but significant role in the investigation of small molecule inhibitors for Alzheimer's disease (AD) by Hong and co-workers described in Brain Research. The discovery of small molecule inhibitors of cytotoxicity induced by amyloid-beta (Abeta) oligomers, applied extracellularly or accumulated intraneuronally, is an important goal of drug development for Alzheimer's disease (AD), but has been limited by the lack of efficient screening methods. The authors use negative stain electron microscopy to characterize the action of inhibitors upon amyloid-beta fibrils and test potential screening assays. For EM, samples of 3 µL were applied to the charged grid and allowed to settle for 10 seconds. The solution was removed by blotting with a piece of filter paper and the sample washed once with 3 µL of water, then stained with a mixture of methylamine tungstate (Nano-W) and 1% trehalose. After 2 seconds, the staining solution was blotted off with a piece of filter paper. The staining procedure was repeated 3 times. Tobacco mosaic virus (TMV) used for internal control and calibration, and samples observed by a transmission electron microscopy. The authors describe two cell-based methods. The first method takes advantage of the unique ability of extracellularly applied Abeta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and employs a short protocol to quantify this toxicity: this method quickly identified two novel inhibitors from two compound libraries. A second, independent screen of the same libraries using a previously reported MC65 protection assay, which identifies inhibitors of toxicity related to intracellular Abeta oligomers, identified the same two leads, suggesting that both assays select for the same anti-Abeta oligomer properties displayed by these compounds. One of the inhibitors, A5, attenuated the progressive aggregation of existing Abeta oligomers, reduced the level of intracellular Abeta oligomers, and prevented the Abeta oligomer-induced death of primary cortical neurons; these results suggest that, when combined, the two methods would generate fewer false results and give a high likelihood of identifying leads with promise for ameliorating Abeta oligomer-induced toxicities at both intraneuronal and extracellular sites. The assays are simple, suitable for rapid screening of a large number of libraries, and amenable for automation.
Two recent papers in Science report the contributions of gold nanoparticles to advances in catalysis and the preparation of nanostructured materials respectively. In the first, Zhang and group demonstrated that platinum (Pt) oxygen-reduction fuel-cell electrocatalysts can be stabilized against dissolution under potential cycling regimes (a continuing problem in catalytic converters used for vehicle exhaust applications) by modifying Pt nanoparticles with gold (Au) clusters a few nanometers in diameter. This behavior was observed under the oxidizing conditions of the oxygen reduction reaction and potential cycling between 0.6 and 1.1 volts in over 30,000 cycles. There were insignificant changes in the activity and surface area of Au-modified Pt over the course of cycling, in contrast to sizable losses observed with the pure platinum catalyst under the same conditions. In situ x-ray absorption near-edge spectroscopy and voltammetry data suggested that the gold clusters confer stability by raising the Pt oxidation potential. Could a new gold standard for mufflers - or at least catalytic converters - be in the offing?
- Hong, H. S.; Maezawa, I.; Yao, N.; Xu, B.; Diaz-Avalos, R.; Rana, S.; Hua, D. H.; Cheng, R. H.; Lam, K. S., and Jin LW.: Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Abeta oligomer-induced cytotoxicity. Brain Res., 1130, 223-234 (2007).
- Zhang, J.; Sasaki, K.; Sutter, E., and Adzic, R. R.: Stabilization of platinum oxygen-reduction electrocatalysts using gold clusters. Science, 315, 220-222 (2007).
In the second publication, DeVries and co-workers report a finding that may have many applications in the construction of nanostructure materials from gold nanoparticles: directionally consistent divalent functionalization of gold nanoparticles. This group placed target molecules specifically at two diametrically opposed positions in the molecular coating of metal nanoparticles using the functionalization of the polar singularities that must form when a curved surface is coated with ordered monolayers, in this case a phase-separated mixture of ligands. Molecules that can be located at these polar defects may be used as chemical handles to form nanoparticle chains that in turn can generate self-standing films. Gold nanoparticles coated with a binary mixture of 1-nonanethiol (NT) and 4-methylbenzenethiol (MBT) were synthesized, and characterized by scanning tunneling microscopy (STM). Ordered rings similar in nature and spacing to others observed previously were found. To place-exchange at the polar defects, the particles were dissolved in a solution containing 40 molar equivalents of 11-mercaptoundecanoic acid (MUA) activated by N-hydroxysuccinimide.
After stirring for 30 minutes, the reaction was rapidly quenched by Sephadex column filtration or precipitation with deionized water. A two-phase "polymerization" reaction, based on the well-known demonstration procedure to synthesize nylon was then conducted by combining an organic (toluene) phase containing the MUA functionalized particles and a water phase containing divalent 1,6-diaminohexane (DAH): electron microscopy showed the formation of chains of nanoparticles, with interparticle spacing consistent with sizes of the ligands and linking molecules. Mixing of nanoparticles of different sizes produced chains of random composition.
- DeVries, G. A.; Brunnbauer, M.; Hu, Y.; Jackson, A. M.; Long, B.; Neltner, B. T.; Uzun, O.; Wunsch, B. H., and Stellacci, F.: Stabilization of platinum oxygen-reduction electrocatalysts using gold clusters. Science, 315, 358-361 (2007).
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .