N A N O P R O B E S     E - N E W S

Jones et al now report the preparation and use of Nanogold®-labeled alpha-bungarotoxin. The conjugate was prepared by using Mono-Sulfo-NHS-Nanogold to label the toxin; conjugation was carried our using standard procedures given in our product instructions, and the product was purified by gel filtration. In order to compare the relative dimensions of alpha-Bgt and Nanogold, the physical size of alpha-Bgt was calculated to using a modeling package; it was shown to be 4.5 times larger than the gold core of the Nanogold particle. Displacement assays showed that alpha-Bgt binding was preserved in the Nanogold conjugate: Ki values are 7.8 and 4.2 nM for gold conjugated alpha-Bgt and unconjugated alpha-Bgt, respectively.

Gold labeled peptides have several advantages for labeling:

• Restricted targets are accessed more easily by small peptide probes.
• Smaller probes mean higher resolution - the gold label is closer to the target.
• Labeling is more simple - no cross-reactivity issues with secondary probes.

Labeling was conducted using a pre-embedding labeling procedure and observed both by light and electron microscopy. At the light microscope level, gold alpha-Bgt binding sites were mostly on the section surface, associated with the cell body and dendritic membranes. In the electron microscope, gold alpha-Bgt binding was observed on, or within a few micrometers, of the section surface. Labeling in the stratum radiatum was observed on dendritic membranes at both synaptic and perisynaptic loci; no cytoplasmic labeling was observed.

Regions of interest were dissected and tissue blocks glued to the stage of a vibrating microtome, submerged in ice cold artificial cerebrospinal fluid (ACSF). 150 micrometer sections were transferred to glass vials containing cold ACSF on a shaker, allowed to reach room temperature, washed 3 x 5 min in ACSF and then blocked in ACSF with 0.2 % acetylated bovine serum albumin (ACSF-BSAc) for 30 min. After rinsing briefly in ACSF, sections were incubated in gold alpha-Bgt (1100 nM) in ACSF-BSAc for 1 h, then washed 3 x 10 min in ACSF-BSAc and fixed in ACSF containing 2.5 % glutaraldehyde for 30 min. Fixed sections were washed 4 x 10 min in deionised water (DW) and silver enhanced for 20 min (for light microscopy; SE-LM reagent, Aurion) or 60 min (for electron microscopy; SE-EM reagent, Aurion). Sections for light microscopy were air-dried onto poly-l-lysine coated microscope slides, defatted by passing through an alcohol series, Nissl stained, dehydrated and mounted in DPX mountant. For electron microscopy, sections were dehydrated through an alcohol series into propylene oxide, infiltrated with epoxy resin overnight, flat embedded on microscope slides then cured in an oven at 60°C for 48 h. Ultrathin sections (60 nm) were collected onto pioloform-coated nickel slot grids and counterstained with uranyl acetate and lead citrate.

Reference:

Jones; I. W.; Barik, J.; O' Neill, M. J., and Wonnacott, S.: Alpha bungarotoxin-1.4 nm gold: a novel conjugate for visualising the precise subcellular distribution of alpha 7* nicotinic acetylcholine receptors. J. Neurosci. Methods, 134, 65-74 (2004).

The sizes that we give for Nanogold® and undecagold refer to the size of the gold core, which is what shows up in the electron microscope. However, in addition to the core, Nanogold and undecagold also contain a layer of small organic molecules on their surface. These solubilize the particles in water, prevent them from adhering non-specifically to biological materials, and also incorporate the reactive group through which the particles are linked to the conjugate biomolecule.

When you are planning labeling reactions, and particularly how you will separate labeled products, it is important to base your plans on the size of the entire label, not just the size of the gold core. The ligand shell is about 0.6 nm thick, and therefore adds about 1.2 nm to the particle diameter. In separation applications, Nanogold will behave very similarly to a globular biomolecule with a diameter of 2.5 to 2.7 nm, while undecagold will behave similarly to a globular biomolecule with a diameter of 2 nm.

Because of the contribution of the heavy gold atoms in the cores of these compounds to their molecular weight, they are smaller than biopolymers of similar molecular weight, and this should be taken into account when deciding how to separate the conjugate. Although Nanogold has a molecular weight of close to 15,000, when it is separated by gel filtration, it will elute similarly to a biomolecule of MW between 7,000 and 10,000. Undecagold, which has a molecular weight close to 5,000, will elute similarly to a 2,500 to 4,000 MW biomolecule.

Concentration of Conjugates:

The concentrations that we give for Nanogold-labeled products are calculated based only on the conjugate biomolecule - the active antibody or streptavidin. They do not include either the mass of the attached gold particle, or any other proteins or macromolecules added as stabilizers or protective agents. All our catalog Nanogold antibody and protein conjugates are supplied at a concentration of 80 micrograms per mL (0.08 mg/mL) of antibody or protein before the weight of the gold label.

How much is that in molecular terms? Our IgG conjugates (MW of IgG = 150,000) contain 0.53 nmol/mL of gold-labeled IgG; our Fab' conjugates (MW of Fab' = 50,000) contain 1.6 nmol/mL of labeled Fab'; and our streptavidin conjugates (MW of streptavidin taken to be 60,000) contain 1.3 nmol/mL of labeled streptavidin.

More information:

• May safely be used before osmium tetroxide - no etching.
• Gold gives a much better backscatter signal than silver for SEM.
• May be used in physiological buffers (chlorides precipitate silver, but not gold).
• Reaction is less pH sensitive than silver
• GoldEnhance is near neutral pH for best ultrastructural preservation.
• low viscosity for easy and accurate mixing of components.

Richards and co-workers illustrate several of these advantages in their publications. Having previously demonstrated the utility of gold enhancement for immunogold staining of cells cultured on metal surfaces, they now report a study of cell focal adhesion using a novel dual labeling method: gold enhanced immunogold, and autoradiographically deposited silver. Electron energy sectioning of the sample, by varying the accelerating voltage of the electron beam, combined with backscattered electron (BSE) imaging, allowed for S-phase cell identification in one energy plane image and quantitation of immunogold label in another. As a result, it was possible simultaneously to identify S-phase cells and their immunogold-labeled focal adhesions sites on the same cell.

S-phase cells were identified autoradiographically by 30 minutes incubation with the equivalent of 4 ACi/ml of tritiated (6-3H) thymidine, placed in the culture medium and mixed thoroughly. The radioactive medium was then chased out with nonradioactive medium for 2 h and the cells were processed for immunolabeling. The cells were rinsed in 0.1 M PIPES buffer, permeabilised in 0.1 % Triton for 1 min, fixed in buffered 4 % paraformaldehyde for 5 min, rinsed again in buffer and then nonspecific antigenic sites were blocked with buffered 1% bovine serum albumin (BSA) and 0.1 % Tween 20 for 15 minutes. Cells were incubated in a solution of mouse anti-human vinculin primary (dilution of 1:300) for 1 h. Non-specific binding sites blocked with 5 % goat serum in buffered 1 % BSA and 0.1 % Tween 20 for 15 minutes; goat anti-mouse 5 nm gold secondary was then applied at 1:200 dilution for 2 h. Cells were fixed permanently with buffered 2.5 % glutaraldehyde for 5 minutes, gold enhanced for 5 minutes, and contrasted with 1% osmium tetroxide, buffered to pH 6.8, for 1 h.

At 8 kV, both the whole cell (autoradiographically deposited silver, and immunogold-labeled focal adhesions were visualized, whereas a lower beam energy of 4 kV was used to visualize exclusively the labeled focal adhesions. The results indicated that cell cycle phase was a significant factor in determining the density of focal adhesions: non-S-phase cells showed a larger adhesion density than S-phase cells. It was therefore shown that influence of cell cycle phases must be considered when quantitation of focal adhesion sites is required.

Reference:

Meredith, D. O.; Owen, G. R.; ap Gwynn, I., and Richards, R. G.: Variation in cell-substratum adhesion in relation to cell cycle phases. Exp. Cell Res., 293, 58-67 (2004).

The samples were characterized by variety of spectroscopic methods. The protection was found to be complete with about 65 and 585 cfH molecules covering the 3-4 and 15-20 nm particles, respectively. Cyclic voltammetry of the free and gold-conjugated showed a shift in the reduction potentials to less negative values upon adsorption to the gold particles; the shifts were similar with 3-4 and 15-20 nm particles. These results show that the nitrogen atom of the NH moiety of piperazine group binds to the gold surface.

Desorption profiles were obtained by dividing a solution of conjugate into aliquots and centrifugation at each of a series of time points, monitoring absorption of the supernatent. A plot of absorption intensity against time gave the desorption profile of cfH. The rate of release from the nanoparticles was found to be faster in bicarbonate solution than in pure water. Kinetics also depended on particle size; faster desorption was seen with the smaller particles. Most of the bound molecules could be released over an extended period of time. This study shows that metal nanoparticles are potentially useful carriers for the delivery and controlled release of cfH and fluoroquinolone molecules.

Reference:

Tom, R. T.; Suryanarayanan, V.; Reddy, G.; Baskaran, S., and Pradeep, T.: Ciprofloxacin-Protected Gold Nanoparticles. Langmuir, 20, 1909-1914 (2004).

Reference:

Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin, X. M.; Jaeger, H., and Lindquist, S. L.: Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition. Proc. Natl. Acad. Sci. USA, 100, 4527-4532 (2003).