NANOPROBES INC.

Detecting Biological Molecules-Nanogold Probes and Peptides

Nanoprobes was founded to develop the most sensitive reagents and technology for detecting biological molecules. Our unique gold labeling technology uses chemically cross-linked metal clusters and nanoparticles as labels. Unlike conventional immunogold probes, in which colloidal gold particles are electrostatically adsorbed to antibodies and proteins, our gold labels are uncharged molecules which are cross-linked to specific sites on biomolecules. This gives our probes a range and versatility which is not available with colloidal gold. Our labels can be attached to any molecule with a reactive group -- proteins, peptides, oligonucleotides, small molecules and lipids -- for detection and localization. Other labels can be combined with our gold labels; our unique FluoroNanogold probes combine Nanogold® and fluorescein into a single probe for imaging a specimen both by fluorescence and electron microscopy. New probes can be engineered based on any fragment of a naturally occurring biomolecule, and the label can be positioned away from the binding site so it does not interfere with binding. Our 1.4 nm Nanogold® probes have been cited in over 80 publications, and we are now developing larger cross-linkable labels in order to offer our customers larger covalently linkable probes with the same advantages.

Have you ever wondered how those medical test kits (the ones that give you a little plus if you're pregnant, a minus if you're not, for example) work? These are one application of the technology in which we specialize: immunogold labeling and immunoassay tests. This section is intended as an introduction to this technology; take our 'virtual technology tour' below to find out about the science behind such products, check out some results from scientists who have used our probes, and see our news update for a complete listing of recent company news.

In Situ Hybridization with Nanogold®-Streptavidin

The small size of Nanogold® probes, their lack of aggregation and their consequent ability to access nuclear antigens makes them an excellent reagent for ultrasensitive in situ hybridization detection of specific DNA sequences both with and without in situ PCR.

Advantages

• More sensitive than NBT-BCIP alkaline phosphatase or peroxidase in combination with label amplification single gene copy resolution.
• No need for the more lengthy PCR procedures for most cases.
• Avoid false positives found with PCR due to mispriming and amplicon diffusion.
• Black color easily seen with standard bright field microscope; no expensive fluorescent optics required, or problems with autofluorescence and loss of signal from bleaching.
• Black color compatible with full strength standard cell and nuclear stains (H & E, nuclear fast red, methyl green) which greatly improves morphological assessment.
• May be used for EM studies as well.

NANOGOLD-PEPTIDES

Several reports have now appeared in the literature describing the covalent attachment of Nanogold® to peptides and their use to track peptide binding both in tissues (using light microscopy) and to individual molecules (using electron microscopy). Retention of activity was demonstrated by Western blots, polyacrylamide gel electophoresis, and competitive binding studies with unlabeled peptide.

How Nanogold-Peptide Probes Are Made
Nanogold® is a small (1.4 nm) gold cluster with an organic shell and a monofunctonal reactive arm, either a maleimide (which reacts with thiols) or a sulfo-N- hydroxysuccinimide ester (which reacts with amines). Covalent linking of the Nanogold to peptides may be achieved by reacting with the alpha-NH2 group, a lysine, a terminal cysteine that is added, or an existing cysteine.

Nanogold® Labeling Reagents, Fluorescent Conjugation, Oligonucleotides, Enzyme Inhibitors

NANOGOLD® LABELING REAGENTS

Nanogold® is a better gold label. The 1.4 nm Nanogold® particle is a gold compound: it is not just adsorbed to proteins, like colloidal gold, but covalently reacts at specific sites under mild buffer conditions. This gives a well defined product that can be purified chromatographically.

Nanogold® brings the versatility of fluorescent conjugation to gold labeling. It may be used to label any molecule with a suitable reactive group: oligonucleotides, lipids, peptides, proteins, enzyme inhibitors and others, unlike colloidal gold which may be adsorbed only to antibodies and a limited range of proteins and peptides. Nanogold® is small and highly uniform in size, in sharp contrast to small colloidal gold preparations (most commonly used "1 nm" golds actually range from 1 to 3 nm). Nanogold® is available both as a labeling reagent for labeling your own biomolecules and in a range of antibody IgG, Fab' and streptavidin conjugates.

Although 1.4 nm Nanogold® is smaller than most other EM probes, it can easily be enhanced with silver (LI Silver or HQ Silver) by brief exposure (1 - 5 min.) to produce highly visible grains 2-20 nm in size (depending on development time). Further development (8-25 min.) gives a black signal easily seen in the light microscope and on immunoblots, polyacrylamide gels and Western blots.

Nanogold® labeling reagents are now available in three different packages: 30 nmol for larger labeling experiments, 5 X 6 nmol for several smaller labeling experiments, or in single 6 nmol vials so you can try these reagents on a smaller scale for a low price.

Features of Nanogold®

• Unparalleled penetration of conjugates up to 40 µm.
• Higher density of immunolabeling than with larger gold probes.
• Can be conjugated to any molecule with a suitable reactive group. Available with different reactivities.
• Extremely uniform 1.4 nm gold particle.
• Label at specific sites which do not obstruct native reactivity.
• Close to stoichiometric labeling.
• Reacts under mild, neutral conditions.
• Conjugates are easily isolated by gel filtration.
• Conjugates are stable to a wide range of pH and ionic strengths.
• High stability: conjugates show unchanged reactivity after storage for a year. 

NANOGOLD®-ANTIBODY CONJUGATES

Nanogold® is a better gold label. The 1.4 nm Nanogold® particle is a gold compound: it is not just adsorbed to proteins, like colloidal gold, but covalently reacts at specific sites under mild buffer conditions.

The Nanogold® particle is covalently and specifically linked to a hinge thiol on Fab' or IgG. The conjugate therefore has excellent stability compared to colloidal gold-antibody preparations. It is the first probe offered as a Fab' conjugate, which is the smallest gold-antibody probe commercially available. These substantially smaller probes reach more antigens and provide better labeling. They can be viewed directly in TEM without silver enhancement or developed with silver to any appropriate size for enhanced visibility with other counterstains. Whereas the stoichiometry of conventional gold probes particles to IgG molecules varies from 0.2 to 10, Nanogold® preparations contain close to one Nanogold® to one Fab' or IgG.

• Smallest commercially available gold immunoprobes.
• Penetrates and reaches antigens inaccessible to other probes: proven penetration up to 40 µm into
• Extremely uniform 1.4 nm diameter gold label.
• Fab', IgG and Streptavidin conjugates available.
• Close to 1 Nanogold® particle to 1 Fab' (or IgG).
• Low non-specific affinity gives minimal background.
• Ultrasensitivity with silver enhancement: 0.1 pg of antigen detection on immunoblots.
• Gold is covalently attached to antibody remote from antigen binding region.
• High stability and long shelf life: conjugates show unchanged reactivity after storage for a year.
• Stable to a wide range of pH and ionic strengths.

Ni-NTA-Nanogold®
Ni-NTA-Nanogold® is designed for detection or localization of polyhistidine (his) -tagged fusion proteins using electron microscopy, light microscopy or blotting. Using Ni-NTA-Nanogold®, his-tagged fusion proteins originating from any of a variety of expression vectors can be labeled under non-denaturing or denaturing conditions. The labeled his-tagged fusion proteins can be visualized by microscope or blots, when used with gold or silver enhancement reagents such as our Gold Enhance EM (Catalog number 2113), Gold Enhance LM (Catalog number 2112), or HQ Silver (Catalog Number 2012). 
Ni-NTA-Nanogold® is designed for detection and localization of polyhistidine-tagged proteins using electron microscopy, light microscopy and blotting. Ni-NTA-Nanogold® comprises a 1.8 nm Nanogold particle with multiple nickel-nitrilotriacetic acid functionalities incorporated into the ligands on the surface of gold particles. Nickel-nitrilotriacetic acid functionalities bind to histidines from the tagged proteins, and form stable complexes with extremely low dissociation constants. Using Ni-NTA-Nanogold®, His-tagged fusion proteins originating from any of a variety of expression vectors can be labeled under both non-denaturing and denaturing conditions. By using silver or gold enhancement, The labeled his-tagged proteins can be visualized in the electron or light microscope and on blots.

Features of Ni-NTA-Nanogold®

• Detects and localizes His-tagged recombinant proteins in electron microscope, light microscope and blots.
• Good product stability.
• 1.8 nm diameter gold particle.
• High resolution for ultrastructural studies.
• Permanent stain does not fade.

Nanogold® Cluster, Photobleaching, Fluorescein, Confocal Microscope, Antibody Fragments

FLUORONANOGOLD

FluoroNanogold is a new immunoprobe which contains both a fluorescent label and the Nanogold® cluster. FluoroNanogold is now available with Alexa Fluor®* 488 or Alexa Fluor®* 594, giving you the benefits of brighter fluorescence, reduced photobleaching, and compatibility with a wider pH range, or in its original formulation with fluorescein as the fluorophore. FluoroNanogold is offered in 1 mL or the more affordable 0.5 mL size

By combining gold and fluorescence into one probe, the same specimen may be imaged using both fluorescence microscopy (e.g., with a confocal microscope) and at the ultrastructural level by electron microscopy. Unlike colloidal golds, which have been found to quench fluorescence, Nanogold® shows little effect. All components are covalently attached to ensure stability and long shelf life. Fab' antibody fragments are much smaller probes than IgG conjugates, and have shown excellent penetration into cells and nuclei. FluoroNanogold conjugates are chromatographically purified to eliminate any aggregates, free gold or unattached fluorescent molecules.

Features of FluoroNanogold

• Unprecedented correlation between fluorescence and EM data.
• Single labeling procedure means less chance for specimen perturbation.
• Choice of new fluorophores for brighter fluorescence, lower background and multicolor labeling.
• Same excellent penetration as found with Nanogold®: much better than 5 and 10 nm colloidal gold probes.
• FluoroNanogold probes are smaller than IgG conjugates.
• Covalent coupling of both labels gives stability and long shelf life.
• Conjugates are stable and fluorescent at a wide range of pH and ionic strengths.

GOLDEN LIPIDS

Gold lipids are another unique product from Nanoprobes. Each lipid molecule is covalently linked to a Nanogold® or undecagold cluster through its polar head group. Since the gold moiety is hydrophilic, these molecules retain the amphiphilic nature of the parent lipid, and behave like fatty acids or phopholipids. They may be used to study membranes by incorporation into the lipid phase, or they may be used to gold-label micelles and vesicles so that they can be identified and visualized. The gold labels may be visualized directly by EM. Using silver enhancement, they may be seen in the light microscope, or by eye in lipoimmunoassays and similar applications.

Features

• Discrete single molecules with one lipid to one gold cluster label.
• Covalent attachment ensures consistent structure and properties.
• Stable, long shelf life.
• Soluble in organic solvents and alcohols for easy incorporation into liposomes.

Silver Enhancement and Amplification: Autometallography

Metal particles can nucleate the highly specific deposition of silver from an appropriate silver salt solution in the presence of a suitable reducing agent. The silver coated gold particle then catalyzes more silver deposition and the silver grains grow in size. In this way even small gold particles such as Nanogold® may be enlarged up to 30 to 100 nm in size, resulting in greatly enhanced visibility by electron microscopy, dark staining in the light microscope and the ultrasensitive visualization of antigens on blots and in gels. Nanoprobes offers two silver enhancers with complementary properties: HQ Silver and LI Silver.

Features of Nanoprobes Silver Enhancers

• HQ Silver
• Unique formulation for the best possible results with Nanogold® in critical applications, particularly in the electron microscope. Slightly light sensitive: should be used in indirect light or with a SafeLight (For a comparison with other silver enhancers, see: Humbel, B. M., et al.: J. Histochem. Cytochem., 43, 735-737 (1995). For uses, see: Baude, A., et al.: Neuroscience, 69, 1031-1055 (1997)).
• Neutral pH for excellent preservation of structural integrity.
• Protective thickening agent for the most uniform, reproducible particle size distribution.
• Low background.
• Compatible with all immunogold reagents.
• Best for electron microscopy.

Microscopists, Gold Particles, Gold Immunoprobe, Goldenhance, Osmium Tetroxide Staining

Gold Enhancement

Silver enhancement has long been used by microscopists to better visualize gold labeling, especially when using small gold particles. Silver is specifically deposited around the gold particle, growing it in size. For TEM, the 1.4 nm Nanogold® can be enlarged to 10-20 nm for clear visiblility, even at low magnifications. Using an initial small gold immunoprobe allows better penetration into tissues (up to 40 microns!) and better labeling of antigens. Then silver enhancement makes everything clearly visible. The development time can be extended to deposit more silver and make the signal visible by bright field (or reflection) light microscopy. With a little more development time, the signal can be seen by the naked eye, and can be used with dot blots or Westerns.

Nanoprobes has now extended this technology by introducing a GOLD developer, "GoldEnhance". It works similarly to silver enhancement, but deposits gold around the initial gold particle.

Why GOLD? There are a number of advantages:

• may safely be used before osmium tetroxide staining (silver is dissolved by the oxidizing agent O4; gold is stable)
• lower backgrounds than silver in some cases; autonucleation minimal even after 1-2 hours
• for SEM, gold gives a much better backscatter signal than silver
• compatible with physiological buffers (silver precipitates with chloride ion, as in PBS buffer; gold does not)
• reaction is less pH senstitive than silver, and GoldEnhance is near neutral for best structural preservation of biological samples (many silver enhancers have a pH of 3-4) excellent shelf life
• low viscosity for easy and accurate mixing of components

Negative Staining

Negative stains are reagents which contain heavy atoms and do not crystallize upon drying, so that they provide a uniform electron-dense stain for electron microscopy. They are used to visualize the edges of protein complexes, macromolecules and cells in suspension. Unlike positive stains such as osmium tetroxide, negative stains do not obscure labeling of the biological structures themselves. Nanoprobes offers two unique, high-quality negative stains: NanoVan and Nano-W.

Conducting Polymers

Polypyrroles are used to form a thin molecular monolayer which is highly conductive (better than carbon); this is an ideal substrate for electron microscopy. To prepare the layer, unsubstituted pyrrole is mixed with one of the Nanoprobes surface active pyrroles (30DP or 30DOP) and applied to a water surface containing ferric chloride. The properties of the film are controlled using Langmuir-Blodgett techniques, and it is then used to coat grids for electron microscope observation.

Undecagold

Undecagold (Au11) is smaller than Nanogold®, with a core of 11 gold atoms only 0.8 nm in diameter. It is ideal for ultra-high-resolution EM work such as scanning transmission electron microscopy, or for resolving elements of large structures by TEM in conjunction with image processing. Undecagold has been used to see the biotin binding sites on avidin to 1 nm resolution by electron microscopy. It is prepared in a form with one reactive arm for cross-linking to a specific site on a target molecule, and is available with different reactivities for labeling different sites.
Note: Single undecagold clusters are not routinely visualized directly in the TEM. Undecagold may be seen upon image processing of protein helices and crystals, or visualized en masse if there is a bulk deposition such as staining of an organelle. Also, undecagold develops more slowly and with less final silver deposition than Nanogold®. Therefore, for many applications we recommend Nanogold®.

Features of Undecagold

• Ultra small 0.8 nm gold core.
• Use to prepare the smallest possible gold probes.
• Highest possible resolution.
• Site specific covalent labeling with choice of reactivities.

Custom Labeling and Custom Synthesis

We are currently able to offer custom labeling with Nanogold® or colloidal gold-labeled antibodies. Nanogold® labeling is restricted to the preparation of labeled Fab; fragments from F(ab')2 fragments, or the labeling of IgG molecules. While we will be glad to consider other requests, our time and resource to undertake such syntheses are limited. Please be advised also that new syntheses frequently require much more work than anticipated, and if a similar procedure has not been demonstrated before, may be better handled as a contract research project or collaboration.

Before requesting a custom synthesis quotation, we recommend that you consider our labeling reagents, which you may use to label a wide variety of molecules. Monomaleimido-Nanogold® reacts slectively with thiols (such as cysteines); Mono-Sulfo-NHS-Nanogold® labels primary alihatic amines (N-terminal or lysine residues) while Monoamino-Nanogold®M can be used with a variety of homo- or heterobifunctional cross-linkers, or directly to label RNA or glycoproteins.

Guidelines

Because the gold cluster labels developed by Nanoprobes, incorporated are attached by covalent cross-linking, they may be used to label almost any molecule with a suitably reactive functionality. We have already conjugated our probes to proteins, lectins, peptides, lipids, biotin and cytoskeletally active probes such as modified phalloidins. Although we are currently only able to complete custom syntheses of Nanogold® or colloidal gold-labeled antibodies, we are pleased to advise on the use of our labeling reagents to label other molecules. Please telephone, fax or E-mail us. Since our researchers are not necessarily familiar with your particular probe or application, we can often provide quotations and suggestions much more quickly if you provide some key information. The following data are particularly useful:

Molecular weight: All our labeling reactions use specific molar ratios of gold to probe. We need the molecular weight to calculate how much gold labeling reagent we will need, how much of the probe we will need to use for labeling, and also which product isolation protocol to use.

Optical density or UV/visible absorbtion data: The extinction coefficients of undecagold, Nanogold® and FluoroNanogold at specific wavelengths have been accurately determined, and these values allow us to determine how successfully your probe has been labeled. If you supply or refer us to specific values for your probe, we can provide you with the exact ratio of gold cluster label to probe in the product.

Biomolecules, HQ Silver Enhancement, Sodium Phosphate Buffer, Phosphate Buffered Saline

Applications of Nanogold® and Related Reagents

Because of its site-specific covalent attachment to biomolecules, and its physical and chemical stability, Nanogold® lends itself to many applications for which colloidal gold is not suitable.

Product applications describe applications of our current products, while research applications describe novel probes and reagents which are not available as products or are under development as future new products.

Product Applications

Following are notes and reports on some unique results obtained using Nanogold® probes:

High-Density, Reliable Pre-Embedding Nanogold® Labeling Procedures
Procedure using HQ Silver Enhancement

• Higher density gold labeling than with other methods.
• Very high specificity.
• Maximum resolution.

This procedure has been described by Tanner and co-workers, and is reported to give significantly higher densities of silver-enhanced gold particles than other methods. An example of the results is shown below:
Materials and Reagents

• Sodium phosphate buffer: 0.1 M sodium phosphate, pH adjusted to 7.4.
• Phosphate-buffered saline (PBS) buffer: 0.02 M sodium phosphate buffer with 0.15 M sodium chloride, pH adjusted to 7.4.
• Phosphate-buffered saline (PBS) buffer: 0.02 M sodium phosphate buffer with 0.15 M sodium chloride, pH adjusted to 7.4, containing (a) 5 % bovine serum albumin and 0.05 to 0.1 % sodium azide; and (b) 1% goat serum and 0.1% NaN3 for 3-4 X 5 min
• Glutaraldehyde and paraformaldehyde.
• HQ Silver reagent (Nanoprobes).
• Deionized or distilled water.

Gold-Based Autometallography

Gold labels such as Nanogold®1 and colloidal gold2 are enlarged and visualized in the electron microscope or optically by the selective deposition of silver onto their surfaces. This process, known as autometallography (AMG), silver amplification3 or silver enhancement,4 is initiated by exposing the particles to a solution containing silver (I) ions and a reducing agent such as hydroquinone2,3 or n-propyl gallate.4 Particles may be enlarged to between 30 and 100 nm in diameter, giving a distinctive black, punctate staining in the light microscope. Nanogold® labeling with silver amplification is one of the most sensitive methods available for histopathology applications such as in situ hybridization. With Catalyzed Reporter Deposition (CARD; also called Tyramide Signal Amplification, or TSA®; NEN Life Sciences, Boston MA), it has been used to detect as few as 1-2 copies of viral DNA or RNA per cell.5 However, its uses are restricted by reactions of silver (I) with halides and other elements in tissues. Also, after signal development, self-nucleation and non-specific background deposition begin quickly, which can make end-point selection difficult or prevent incorporation into automated procedures.

We have found that gold can also be used as an autometallographic agent. In a suitable chemical environment, it may be selectively deposited onto Nanogold® or colloidal gold to generate a high contrast signal in the electron microscope, and black, punctate staining in the light microscope. It is also effective on blots. We find background staining to be equal to or lower than that found with silver enhancement in several test systems; in addition, although development is complete within 20 minutes, autonucleation is minimal even after two hours exposure to the reagent. This gives the gold autometallographic process a potential advantage for automation. In preliminary electron microscope experiments on the in situ detection of DNA and RNA and the labeling of CD44 in astrocytoma cells,6 particle sizes were found to be more uniform than those found using silver enhancement.

Using gold rather than silver has additional advantages. It can be safely used with osmium tetroxide and uranyl acetate staining, conditions which can etch silver-enhanced gold particles, without the need for protective gold toning. The gold autometallographic reaction is tolerant of a wider range of anions, and may be used in physiological buffers (even with chlorides, which precipitate silver; however, water washes are recommended). In the SEM, gold gives far superior backscatter detection compared with silver. Furthermore, the reaction is less pH sensitive than silver enhancement: the formulation is near neutral, which for some tissues is preferable to the low pH (~3.8) of many silver developers for morphological preservation. In in situ hybridization experiments, such as the detection of HPV-16 viral DNA in SiHa cells using CARD with Nanogold®-streptavidin detection, we find that gold autometallography to be as sensitive as silver amplification, with cleaner backgrounds (Figs. 1-4).

The gold autometallography reagent is prepared from three stable components mixed in equal volume, and applied to specimens for times from five minutes to twenty minutes. A "stop" treatment with 1-2 % sodium thiosulfate for 2 minutes was found to produce cleaner backgrounds in the in situ studies, although a simple water wash to stop development has also been used successfully.

Detection of Nanogold®-labeled molecules on Gels

Specific: Develops only Nanogold®-labeled band
Rapid: 1-5 minutes
Sensitive: More sensitive than usual gel stains
May be used directly on gels or on blot transfers

1.4 nm Nanogold® particles may be developed with silver so that they become visible to the naked eye, thus amplifying the signal thousands of times. If you have used monomaleimido-Nangold, mono-NHS-Nanogold® or mono amino-Nanogold® to label a protein or other molecule, these may then easily be analyzed and detected on gels using silver enhancement.

Enzymatic Metallography, Immunohistochemical, In Situ Hybridization, Brightfield Light Microscopy

Research Applications

Enzymatic Metallography: A Simple New Staining Method Enzyme metallography is a simple, new method for immunohistochemical and in situ hybridization staining and detection, in which a targeted enzyme conjugate is used to deposit metal from solution, giving a dense, black, highly resolved signal for brightfield light microscopy and for electron microscopy.

Nickel-NTA-Nanogold Binds his-Tagged Proteins

Six histidines added to expressed proteins have been a boon for rapidly purifying them from the expression organism lysate, since it was found that the 6x-His tag specifically binds (reversibly) to columns containing Ni+2 [1]. The nickel is chelated to the column with nitrilotriacetic acid (NTA), which is similar to EDTA. Since many proteins now have His-tags, and cells can be transfected to produce His-tagged proteins, it is of interest to have a gold label that also binds specifically to this tag. One application is identifying a specific protein against a background, for example in cell sections to see the localization of an expressed protein, much like green fluorescent protein; however, the gold label is visible in the EM for higher resolution studies; it can also be silver or gold enhanced to visualize it in the light microscope, or on blots. Another application is to use it as a molecular domain or subunit label for high resolution single particle analysis. Here, since the position of the His-tag is known, e.g., the amino terminus, that part of the molecule can be locally labeled and useful for cryoEM reconstructions. It would also serve as a high visibility fiducial mark for orienting single particles at low dose, perhaps permitting extension of the resolution obtainable by such techniques. For multi-subunit complexes, a particular subunit may be labeled to specifically identify it in the complex. In x-ray or electron protein crystallography, the label could be used to improve contrast or be used as a phasing aid. Another difference in this type of label is that the Ni-NTA group is quite small compared to an antibody, thus bringing the gold particle much closer to the 6x-His-tag giving higher resolution labeling with less ambiguity or floppiness. The smaller size will also improve its diffusion into cells or tissues.

Two papers have appeared using Ni-NTA-Nanogold [2,3]. These were based on first generation constructs. Several potential problems may occur with this type of probe. Since each Ni atom can only bind two histidines (even in column use), there should be three Ni-NTA groups per gold to fully complex a 6x-His tag and provide maximum affinity. Preparing a gold particle with exactly 3 Ni-NTA groups in the optimal conformation is a challenge and if additional groups are present, this can lead to aggregation of His-tagged proteins in solution, which was observed in some cases. Reducing the number of Ni groups lowers the gold’s affinity. The linker arm from the gold to the NTA group may also be varied to find the optimal structure. A number of different Ni-NTA-gold cluster constructs were therefore synthesized varying the linker and the number of NTA groups. Although further work is in progress, some of these preparations have produced high binding to His-tagged proteins in solution without aggregation. Control samples were the same protein, but without the 6x-histidine residues, and these showed virtually no labeling.

Aldehyde Gold Clusters for Molecular Labeling

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 a-amino1. With some enzymes, catalytic activity after fixing is preserved2; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine3. It has also been reported that glutaraldehyde can polymerize in aqueous solution4. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates5. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.

Gold Cluster Crystals

Gold clusters are gold compounds with a core of gold atoms and organic groups covalently bound to the surface gold atoms. An example is undecagold, Au11(P(C6H5)3)7, whose structure was solved by x-ray crystallography using 3-dimensional crystals. These differ from colloidal gold, which are suspensions of metal particles, usually formed by metal ion reduction; although the particles may be approximately the same size, they vary due to the statistical process of formation. Gold clusters are compounds with a definite formula, and should all be perfectly identical. However, it is known that there is a family of stable gold cluster compounds, such as Au6, Au11, Au13, Au55, Au67, etc. In a given preparation of gold clusters, there is usually some mixture of these, thus leading to some size variation. Methods such as gel filtration column chromatography and ultrafiltration can be used to separate most of these species, so that relatively pure preparations may be achieved. The UV-Vis spectra of the different clusters are also usually significantly different, aiding in identification of the various clusters. Since the larger nuclearity clusters have not been crystallized, their exact structure has not been determined.