Updated: June 14, 2004
N A N O P R O B E S E - N E W S
Vol. 5, No. 6 June 14, 2004
In this Issue:
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This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.
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Because it is a molecular label, Nanogold® provides both higher and more consistent resolution than colloidal gold probes. There are two reasons for higher resolution: Nanogold is coated only with a very thin layer of small organic molecules, not with the proteins and macromolecules required to stabilize colloidal gold, and therefore the entire gold probe is smaller than a colloidal gold conjugate; and because it is covalently linked, Nanogold may be conjugated to smaller molecules than colloidal gold. Undecagold, which has the same advantages, has been used to label other small molecules, and most molecules that have been labeled with undecagold may be labeled with Nanogold in the same manner.
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Examples of molecules that have been successfully labeled include:
Several papers have demonstrated the improved resolution available through covalent cluster labeling. For examples, see:
- Hainfeld, J.F. and Furuya, F.R. A 1.4nm Gold cluster covalently attached to antibodies improves immunolabeling, J. Histochem. Cytochem., 40, 177-184 (1992).
- Hainfeld, J.F. Undecagold-antibody method. In Colloidal Gold: Principles, Methods, and Applications., M. A. Hayat (Ed.), San Diego, Academic Press; Vol. 2, pp. 413-429 (1989).
You can also increase resolution by decreasing the number of layers between the target and the gold probe, by labeling a small-molecule primary probe directly with gold instead of localizing it with a much larger gold-labeled antibody. As well as using smaller fragments of antibodies and proteins, Nanogold and undecagold can also be cross-linked to molecules that are difficult or impossible to label with colloidal gold, such as lipids, or oligonucleotides.
Nanogold and undecagold labeling are site-specific: hence, a molecule labeled at a unique functional group will always be labeled at the same site. For example, antibodies reduced using the appropriate conditions and conjugated with Monomaleimido Nanogold will always be labeled at the hinge region, as demonstrated by Hainfeld and Furuya in the reference above.
More information:
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Paspalas and Goldman-Rakic have describe a novel double labeling method in which two antigens were distinguished by Nanogold labeling followed by gold enhancement for different times to give two different, non-overlapping size distributions. Using this method, dopamine D5 (but not D1 or D2) receptors in the perisomatic plasma membrane of prefrontal cortical neurons were found to form discrete, exclusively extrasynaptic microdomains with inositol 1,4,5-trisphosphate-gated calcium stores of subsurface cisterns and mitochondria (localized using the 1,4,5-trisphosphate receptor (InsP3R)). These findings indicate that a novel dopaminoceptive substratum is present in the brain, and that a unique D5 receptor-specific signaling paradigm exists.
Brains from two adult rhesus monkeys, anesthetized with sodium pentobarbital (100 mg/kg, i.v.), and perfused transcardially with oxygenated artificial CSF followed by 4% paraformaldehyde / 0.08% glutaraldehyde in phosphate buffer (PB; 100mM) and aldehyde-free PB, were blocked coronally, vibrosliced at 60 microns, cryoprotected, and stored frozen at -80°C. Prefrontal cortical sections were thawed in PB and preincubated for 45 min in 10% normal goat serum and 2% bovine serum albumin in Tris-buffered saline (TBS; 50mM) before being transferred to primary antibodies.
Primary antibodies in N-TBS were simultaneously applied for
48 hr at 4°C. To visualize D5R, sections were preincubated for 30 min in N-TBS supplemented with 0.1% acetylated BSA, 0.1% fish skin gelatin and 0.07% Tween 20 (gold buffer), then incubated for 3 hr in Nanogold-conjugated goat anti-rabbit Fab' (1:200 in gold buffer). After washing in ultrapure water and 20 mM sodium citrate, sections were enhanced for 12 min on ice with GoldEnhance. Subsequently, Nanogold-conjugated goat anti-mouse Fab' (1:200) was applied for 4 hr to probe the InsP3R primary antibody. Sections were finally postfixed in glutaraldehyde and transferred for 4 min to the gold developer to enhance the InsP3R-bound Nanogold but also to enhance further (i.e., 12 + 4 min in total) the D5R gold signal of the first series (two-step autometallography). This sequential enhancement produced distinct, nonoverlapping particle-size groups (see Methodological Considerations). As am alternative method and to provide a control, D5Rs were labeled in a second series by using species-specific bridging antibodies and peroxidaseantiperoxidase tertiary complexes (1:200 in N-TBS for 2 hr each), visualized with 0.025% diaminobenzidine (DAB) in TBS with the addition of 0.007% hydrogen peroxide. InsP3Rs were labeled as detailed above, with gold enhancement ranging from 3 to 8 min at 22°C.
For electron microscopic observation, sections were postfixed in 1% buffered osmium tetroxide (15 and 30 min for silver- and gold-enhanced material, respectively), treated with ethanolic uranyl acetate en block, embedded in Durcupan epoxy resin and polymerized
at 58°C for 48 hr under vacuum. Layers II-IV and V-VI of the dorsolateral PFC (Walkers area 46) were sampled for thin sectioning and ultrastructural analysis using a JEM-1010 transmission electron microscope operated at 80 KV, with or without lead counterstaining.
Reference:
Paspalas, C. D., and Goldman-Rakic, P. S.: Microdomains for dopamine volume neurotransmission in primate prefrontal cortex. J. Neurosci., 24, 5292-5300 (2004).
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We are frequently asked about the solubility of Nanogold®, and how best to label molecules that are soluble in solvents other than water or aqueous buffers, such as peptides, lipids, drugs or other small organic molecules. Nanogold is also soluble in a number of organic solvents, including those that are well-tolerated by biological molecules, and including these in the reaction mixture can increase solution of the reactive species and improve labeling.
Nanogold is soluble in water and in most aqueous buffers, in most cases to at least 1,000 nmol/mL, providing a solution that is concentrated enough for most labeling experiments. However, solution may require gentle agitation for a short time before the reagent dissolves completely.
Nanogold is highly soluble in dimethylsulfoxide (DMSO). Dissolving it in a small quantity of DMSO before adding water will usually speed up solution and ensure an effective reaction. Up to 20% DMSO is also well tolerated by many biological molecules, and therefore we recommend predissolving Nanogold reagents in a small quantity of DMSO (10% of the final reaction volume) before adding water. It is also highly soluble in mixtures of isopropanol, another organic solvent that is well tolerated by many biologicals, in water, so predissolving in 10% isopropanol is a good alternative if DMSO is not suitable for your application. Nanogold is not compatible with N,N-dimethylformamide (DMF): however, in many applications you can use N,N-dimethylacetamide (DMA), which has very similar properties, instead, and Nanogold is soluble and stable in this solvent. Nanogold is also soluble in acetonitrile.
Nanogold is soluble in alcohols, especially ethanol, although it is more soluble in alcohol-water mixtures than in alcohols alone. If you are using ethanol precipitation for a Nanogold-labeled oligonucleotide, then degree to which the labeled oligonucleotide will be precipitated will vary depending on the labeling stoichiometry and the length (and hence contribution to the solubility properties of the conjugate) of the oligonucleotide. Ethanol precipitation alone may give inadequate separation of Nanogold-labeled oligonucleotides, so we recommend that chromatographic separation (reverse-phase, gel filtration and hydrophobic interaction) and UV/visible spectroscopy are used in addition to ethanol precipitation to separate and characterize the reaction products in each phase of the reaction mixture. For a more detailed discussion of oligonucleotide labeling, see last months newsletter.
If you are labeling lipids or other hydrophobic entities, Nanogold is soluble in mixtures of up to 50% dichloromethane (methylene chloride) or trichloromethane (Chloroform) with alcohols, and these should sufficiently solubilize both lipids and Nanogold to allow reaction. Addition of a small amount of an organic-soluble base, such as triethylamine, may help the reaction of Mono-Sulfo-NHS-Nanogold under these conditions but make sure that your base does not contain primary or secondary amines that may react.
Solvent is also a consideration in labeling surfaces. Here, the effect that the solvent-surface interaction has on the ability of the Nanogold to access the surface group must be considered. If you use a solvent that does not wet the surface sufficiently (such as aqueous buffers with hydrophobic polymer surfaces) the gold particles may be hindered from approaching the surface, and adding an organic solvent such as DMSO to enhance wetting will facilitate reaction.
More information:
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Two recent references on the use of gold particles for surface plasmon resonance detection had us wondering, since both mention "Nanogold." However, they mention it incorrectly, using it to describe 20nm and 16nm colloidal gold respectively. Please note that "Nanogold" is a trademark of Nanoprobes, Incorporated, and should not be used to describe colloidal gold. The results, though, suggest some interesting new uses for the real Nanogold.
Surface Plasmon Resonance (SPR) refers to the 520nm peak in the UV/visible spectrum of colloidal gold particles. Changes in the environment of the gold particles, the nature of the surface binding, or the degree of interaction between gold particles, can change this feature, and these changes form the basis for a method to detect binding and changes in biological molecules. Aslan, Lakowicz, and Geddes use the red shift in the resonance wavelength arising from the close proximity of two nanoparticles for glucose sensing, based on the aggregation and disassociation of 20 nm gold particles and the changes in plasmon absorption induced by the presence of glucose. High-molecular-weight dextran-coated nanoparticles are aggregated with concanavalin A (Con A), which results in a significant shift and broadening of the gold plasmon absorption. When glucose is added, it competitively binds to Con A, reducing gold nanoparticle aggregation and therefore the amount of plasmon absorption monitored at a near-red wavelength.
Dextran (average molecular weight: 64,000, 170,000, and 505,000) was immobilized on gold nanoparticles stabilized with a chemisorbed long-chain carboxyl-terminated alkane thiol. The surface carboxyl groups were activated using EDC and NHS and reacted with 2-(2-aminoethoxy)
ethanol (AEE), then the resulting hydroxyl groups were activated using epicholorohydrin and covalently coupled to dextran.
By modifying the amount of dextran or Con A used in the nanoparticle fabrication, it was found that the glucose response range could be adjusted, suggesting that a single sensing platform may potentially be used to monitor micromolar to minimolar glucose levels in different physiological fluids such as tears, blood, and urine.
Reference:
Aslan, K.; Lakowicz, J. R., and Geddes, C. D.: Nanogold-plasmon-resonance-based glucose sensing. Anal. Biochem, 330, 145-155 (2004).
Meanwhile, Hsu and Huang propose a novel amplification method for use on biochips, depositing multiple gold particles at target sites: rolling circle amplification of an oligonucleotide linked to a detection antibody, followed by detection with a gold nanoparticle-linked complementary oligonucleotide. The rolling circle amplification, which is based on a circular template, produces a chain of repeats of the target oligonuceotide linked to the bound antibody. As the authors demonstrate, this results in the deposition of densely aggregated gold nanoparticles at the target, which may then be readily detected optically. This offers the potential of simple optics, combined with higher sensitivity and higher chip density than current fluorescence-based methods.
Nanogold® has already been shown to provide advantages for chip detection, and the addition of silver or gold enhancement may provide substantially improved sensitivity. We have discussed papers by Alexandre, which describe such an experiment, and other applications of gold nanoparticles to biochips in previous issues of this newsletter.
Reference:
Hsu, H.-Y., and Huang, Y.-Y.: RCA combined nanoparticle-based optical detection technique for protein microarray: a novel approach. Biosens. Bioelectron., 30, 123-126 (2004).
More information:
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Some preliminary results using our novel enzyme metallographic detection and staining procedure for electron microscopy will be presented at Microscopy and Microanalysis 2004. Our paper, number 815, entitled "Enzymatic Metallography as a Correlative Light and Electron Microscopy Method" will be given as a platform presentation in session A07, "Biological Specimen Preparation and Labeling," scheduled for Monday 8/2/2004 from 9 AM to 12 noon in room 103 of the Savannah Convention Center.
More information:
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Brolo and group report that a periodic square array of sub-wavelength holes on gold films can be utilized as a SPR sensor; this new sensor also operates in transmission mode, allowing for simpler optics and a smaller probing area. Arrays of 200 nm nanoholes in a 100 nm thick gold film on a glass surface were used to monitor the binding of organic and biological molecules to the metallic surface. This technique is particularly sensitive to surface binding events: sensitivity was found to be 400 nm per refractive index unit, comparable to other grating-based surface plasmon resonance (SPR) devices.
Reference:
Brolo, A. G.; Gordon, R.; Leathem, B., and Kavanagh, K. L.: Surface Plasmon Sensor Based on the Enhanced Light Transmission through Arrays of Nanoholes in Gold Films. Langmuir, 20, 4813-4815 (2004).
Wei and co-workers describe the synthesis of high-aspect gold nanorods from 3-5 nm gold nanoparticles in another paper in Langmuir. Gold nanorods are one-dimensional structures with potentially important size-dependent optical properties and the ability to enhance surface Raman scattering and fluorescence signals. Nanorods were synthesized directly on glass surfaces using seed-mediated deposition of Au from AuCl4- onto surface-attached 3-5 nm diameter gold nanoparticles in the presence of cetyltrimethylammonium bromide (CTAB). Average lengths (200 nm to 1.2 microns) and aspect ratio (6-22) were found to increase with increasing AuCl4- concentration.
Reference:
Wei, Z.; A. Mieszawska, J., and Zamborini, F. P.: Synthesis and Manipulation of High Aspect Ratio Gold Nanorods Grown Directly on Surfaces. Langmuir, 20, 4322-4326 (2004).
If you want platinum rather than gold nanoparticles, your choices are increased by Huang and co-workers, who describe a new method for preparing colloidal platinum particles from the Karstedt catalyst in toluene with PEG and a hydrosilyl precursor. The resulting platinum nanoparticles are water-soluble, stable for several months, and have a mean diameter of 3.0 ± 0.6 nm.
Reference:
Huang, J.; He, C.; Liu, X.; Xiao, Y.; Mya, K. Y., and Chai, J.: Formation and Characterization of Water-Soluble Platinum Nanoparticles Using a Unique Approach Based on the Hydrosilylation Reaction. Langmuir, 20, 5145-5148 (2004).
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