Updated: April 4, 2003
N A N O P R O B E S E - N E W S
Vol. 4, No. 4 April 4, 2003
This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.
Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing tech@nanoprobes.com.
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The applications of Nanogold® and other small metal nanoparticles to nanoelectronics was illustrated again by a recent report from Xiao and co-workers. An apolipoprotein (apo)flavoenzyme, apoglucose oxidase, was reconstituted on a 1.4-nanometer gold nanocrystal (Nanogold) functionalized with the cofactor flavin adenine dinucleotide and integrated into a conductive film. This yielded a bioelectrocatalytic system with exceptional electrical contact with the electrode support, producing an electron transfer turnover rate of the reconstituted bioelectrocatalyst is ~5000 per second, about seven times faster than the rate at which molecular oxygen, the natural cosubstrate of the enzyme, accepts electrons. Nanogold acts as an electron relay or electrical nanoplug for the alignment of the enzyme on the conductive support and for the electrical wiring of its redox-active center.
Two routes were used to construct the reconstituted Au-NP-GOxmonolayer electrode. In the first, Mono-Sulfo-NHS-Nanogold was reacted with N6-(2-aminoethyl)-flavin adenine dinucleotide (FAD) to yield FAD-Nanogold. Apoglucose oxidase (apo-GOx) was then reconstituted with the FAD-functionalized Au-NP. The Nanogoldreconstituted GOx was assembled on a gold electrode functionalized with a 1,4-dimercaptoxylene monolayer. In the second, FAD-functionalized Nanogolds were assembled on the 1,4-dimercaptoxylene-functionalized electrode, and apo-GOx was then assembled on the FAD. Microgravimetric quartz crystal microbalance (QCM) measurements indicated that the surface coverage of glucose ocidase reconstituted onto the Nanogold was found to be 1 X 10-12 mole.cm-2. The affinity of the gold particle for the thiol-functionalized electrode surface, combined with its site-elective binding to the enzyme, acted to orient the enzyme in a position that maximized electron transfer.
Reference:
Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F., and Willner, I.: Plugging into Enzymes: Nanowiring of Redox Enzymes by a Gold Nanoparticle. Science, 299, 1877-1881 (2003).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12649477&dopt=Abstract
More information:
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Richards and co-workers have continued their pioneering work using SEM studies with immunogold labeling and gold enhancement to localize the components that contribute to cell adhesion to different substrates. GoldEnhance uniquely enables these studies: they cannot be achieved with conventional silver enhancement both because the silver enhancement reagents are precipitated by some substrates,and because they are etched by the postifxation conditions used. Recently, they reported a new combination of autoradiography and immunolabelling techniques that allows the simultaneous identification of both S-phase cells and their focal adhesions using scanning electron microscopy.
S-phase cells were radiolabelled with a pulse of tritiated thymidine, which is selectively incorporated into synthesizing DNA. After blocking with 1% bovine serum albumin (BSA) and 0.1% Tween 20 for 15 min, the cells were stained for vinculin by incubation in a solution of mouse anti-human vinculin primary (clone hVin-1; SIGMA: 1 : 300 dilution, 1 h), followed by blocking with 5% goat serum buffered 1% BSA and 0.1% Tween 20 for 15 min, then labeling with a secondary 5 nm gold-goat anti-mouse conjugate (1 : 200 dilution, 2 h). Cells were then fixed permanently with buffered 2.5% glutaraldehyde. Enlargement of the gold probes was performed by enhancement with GoldEnhance EM for 5 minutes; additional contrasting of the cell was accomplished by incubating the cells with 1% osmium tetroxide. The cells were dehydrated, critical point dried, mounted on SEM stubs and coated with a thin layer of carbon (4 nm) before application of nuclear emulsion and exposure. After exposure and development, the cells were dehydrated again and embedded in LR White resin, which was then polymerized before removing the substrate, to expose the embedded cell undersurface. Electron-energy sectioning of the sample by varying the accelerating voltage of the electron beam allowed separate S-phase cell identification in one electron energy section and visualization of immunogold label in another section, within the same cell. This technique enabled the positively identification of S-phase cells and immunogold-labelled focal adhesions on the same cell simultaneously, and thus could be used to quantify focal adhesion sites on different substrates.
Reference:
Owen, G. R.; Meredith, D. O.; ap Gwynn, I., and Richards, R. G.: Simultaneously identifying S-phase labelled cells and immunogold-labelling of vinculin in focal adhesions. J. Microsc., 207, 27-36 (2002).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12135456&dopt=Abstract
Baxter, Richards and co-workers then used this method to evaluate osteoblast and fibroblast adhesion to different substrates, as part of a project to find methods for improving osseointegration at implant/bone interfaces and reducing implant loosening. Fibroblast and osteoblast morphologies and adhesion to various substrates (Thermanox plastic and commercially pure titanium treated using conventional anodisation, plasma spraying of HA, and anodic plasma-chemical (APC) treatment in an electrolyte solution containing either calcium and phosphate (APCCaP) or phosphoric acid (APC-P)) were evaluated using qualitative and quantitative methods. Vinculin was localized as described above using mouse anti-human vinculin primary (clone hVin-1; SIGMA: 1 : 300 dilution, 1 h) and 5 nm gold-goat anti-mouse secondary (1 : 200 dilution, 2 h), followed by gold enhancement (GoldEnhance EM) for 7 minutes. Cells were post-fixed with 1 % osmium tetroxide in PIPES buffer for 1 hour. Specimens were examined using a Hitachi s-4700 field emission SEM fitted with an Autrata yttrium aluminium garnet (YAG) backscattered electron (BSE) detector, operated in HC (high current)-BSE detection mode.
Upon comparison of cell morphology with image analysis to determine the area of immunogold labeling, both osteoblasts and fibroblasts showed extensive cell spreading, total cell area and greatest amount of adhesion, with defined adhesion patterns on the Thermanox plastic, anodised titanium, and the two APC-CaP substrates. With fibroblasts, almost no cell spreading and very low adhesion, was observed in cells cultured on the APC-P and HA surfaces. The extent of cell spreading correlated with the area of focal adhesions as assessed by the amount of vinculin labelling. The Thermanox plastic, anodised titanium, and the two APCCaP substrates were found to be the most cytocompatible substrates.
Reference:
Baxter, L. C.; Frauchiger, V.; Textor, M.; ap Gwynn, I., and Richards, R. G.: Fibroblast and osteoblast adhesion and morphology on calcium phosphate surfaces. Eur. Cell Mat., 4, 1-17 (2002).
Abstract (European Cells and Materials; requires free registration):
http://www.eurocellmat.org.uk/journal/papers/vol004a01.htm
The method is described in detail in this original publication:
Owen, G. R.; Meredith, D. O.; ap Gwynn, I., and Richards, R., G.: Enhancement of immunogold-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions. Cell. Biol. Int., 25, 1251-1259 (2001).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11748918&dopt=Abstract
More information:
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If you'd like to avoid problems with your samples charging under the electron beam, consider our conductive polymer substrates, prepared with 3-octadecylpyrrole and 3-octadecanoylpyrrole. These are used with ferric chloride as a catalyst and a trace of pyrrole vapor to form thin layers of conductive polypyrroles in a Langmuir-Blodgett trough or similar apparatus, which can then be transferred to EM specimens. The resulting films are very uniform, and effectively disperse charge, enabling higher beam current and resolution.
Currently, we do not manufacture, process, or characterize conductive polymers for bulk or industrial applications. If you are interested in this field, the links below may provide more helpful information:
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If you are new to our newsletter and want to catch up on past issues, visit our new archive page, which includes a content list and links to all the articles in past issues. Applications of our current products, and preliminary reports of new technology we are researching and developing, are described in more detail on our Applications page.
More information:
We also welcome Abhishek Bhatnagar, who joined our staff recently as a Research Intern to help with a new project to develop novel probes for the correlative fluorescence and electron microscopic imaging of live cells.
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Differences in the effects of different fixation conditions upon beta-galactosidase (beta-Gal) activity are further explored in the current issue of the Journal of Histochemistry and Cytochemistry. Previously, Ma and group had evaluated the effects of four different fixatives on beta-Gal activity in kidneys from LacZ-stop-human alkaline phosphatase (Z/AP) double reporter mice, and demonstrated that 0.2% glutaraldehyde solution produced strong 5-bromo-4-chloro-3-indolyl-beta-d-galactosidase (X-Gal) staining at room temperature (RT) for 48 hr, while X-Gal staining with 4% paraformaldehyde or 10% formalin fixative was markedly diminished at RT for 48 hr:
Reference:
Ma, W.; Rogers, K.; Zbar, B., and Schmidt, L.: Different fixatives on beta-galactosidase activity. J. Histochem. Cytochem., 50, 14211424 (2002).
Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/10/1421
Takahashi and co-workers, however, find that fixation with 4% paraformaldehyde showed no X-Gal staining at RT at any time point; the differences between their findings and those of Ma et al. might be due to differences in the fixation process (i.e., fixation was performed after, rather than before, tissues were frozen). 100% acetone produced stronger X-Gal staining than 0.2% glutaraldehyde (Figure 1C), suggesting that 100% acetone was effective as fixative for a period of less than 8 hr, even at RT.
Reference:
Takahashi, M.; Hakamata, Y.; Takeuchi, K., and Kobayashi, E.: Effects of Different Fixatives on -galactosidase Activity. J. Histochem. Cytochem., 51, 553-554 (2003).
Schmidt and co-workers describe a detailed study of the expression, characterization and localization of a new desmosomal plaque protein, Plakophilin 3 (PKP3). By immunofluorescence microscopy, PKP3 was localized to desmosomes of most simple and almost all stratified epithelia and cell lines derived from them, with the remarkable exception of hepatocytes and hepatocellular carcinoma cells, and localization was then studied in subcelular detail by electron microscopy using Nanogold® with HQ silver enhancement. For immunoelectron microscopy, cultured MCF-7 cells grown on coverslips were fixed with 2% formaldehyde in PBS for 5 min, permeabilized by incubation with PBS with 0.1% [v/v] saponin for 2 min and incubated with primary antibodies against PKP3 or desmoplakin (DpI/II, multi-epitope cocktail; Progen) usually for at least 3 h, then Nanogold or 5 nm gold-labeled secondary anti-guinea pig or anti-mouse IgG applied overnight, followed by silver enhancement for 3 min.
Reference:
Schmidt, A.; Langbein, L.; Pratzel, S.; Rode, M.; Rackwitz, H. R., and Franke, WW.: Plakophilin 3--a novel cell-type-specific desmosomal plaque protein. Differentiation, 64, 291-306 (1999).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10374265&dopt=Abstract
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