N A N O P R O B E S     E - N E W S

• Abstract (courtesy of PubMed (Medline)).

More information:

• Nanogold reagents: Catalog information
• Monomaleimido Nanogold: Product information
• Mono-Sulfo-NHS-Nanogold: Product information
• Monoamino Nanogold: Product information
• Nanogold technical help: Nucleotide labeling
• References: Nanogold labeling of nucleotides
• DNA Nanowires: our paper from Microscopy & Microanalysis 2001

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"Help, it's Stuck on the Column!" Rescue Bad Nanogold® Separations

Help, my conjugate has crashed out and is stuck on the column! Can we recover it?

The place to look for a solution is in the solubility of Nanogold and its conjugates. Solvents and buffers that dissolve Nanogold, or your conjugate, effectively are more likely to re-solubilize it from column materials. If you encounter this problem, we recommend trying the following:

• 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. Because up to 20% DMSO is also well tolerated by many biological molecules, eluting the column with a buffer containing 20% DMSO may solubilize the conjugate without compromising the reactivity of the conjugate biomolecule. Collect the Nanogold-containing fractions, concentrate again, and repeat the chromatographic separation with 20% DMSO in the eluting buffer.

• Nanogold is also soluble in alcohols and alcohol-water mixtures. Isopropanol and ethanol are well-tolerated by many biological molecules are may therefore be used in up to 20% concentration in the eluting buffer to help redissolve precipitated Nanogold. Using 20% or lower concentration of organic solvent should help to ensure that the conjugate biomolecule will be intact and the conjugate will still bind to its target.

• Addition of a detergent, such as 0.1% Tween-20, may help solubilize Nanogold. Many of the non-specific interactions of Nanogold conjugates with column materials and with biological systems may be attributable to hydrophobic interactions: Tween-20 will help to break these interactions and solubilize the hydrophobic species in aqueous buffers.

• If using 20% organic solvent does not work, stronger measures may be needed. Try injecting 5% of the column volume of pure DMSO: you should see this displace the Nanogold (which will be visible as a brown stain) as the DMSO works its way down the column.

What does a good separation look like?

For an example of gel filtration, check the section on separating your conjugate in our Guide to Gold Cluster Labeling. In the example given, a Nanogold-labeled antibody is separated from excess unconjugated Nanogold. In this case, you see two bands elute: the larger Nanogold-labeled protein is eluted first, and usually appears light brown, while the unbound excess Nanogold elutes later, usually appearing darker brown. The labeling reaction mixture almost always contains some smaller molecules (cross-linkers, buffer components) which elute as a third, usually colorless peak, close to the column volume. If you are labeling a smaller molecule and use an excess of the molecule to be labeled, the Nanogold conjugate will be the first, colored peak to elute; it will be followed by a larger, less colored peak which is excess, unconjugated biomolecule.

The attempt to rescue the conjugate didn't work: now how do I clean the column?

The usual cleaning procedures that are recommended for gel filtration columns are meant for proteins and other types of biological molecules, which these columns are usually used to separate. Gold deposits, however, have different solubility properties, and therefore need different treatments. If you have persistent gold deposits after regular cleaning - particularly the purple or blue color that indicates colloidal gold particles - we suggest the following methods to remove them. Please note that these suggestions may not apply to all columns: you should not use any of these procedures if the instructions for your column caution against it.

• Use a thiol-containing compound - thiols have a high affinity for gold and break down gold deposits and remove them from the column. If you combine this with dimethylsulfoxide (DMSO), which is an excellent solvent for many gold compounds, the result is a highly effective gold removing wash.

  50 or 100 mM mercaptoethylamine hydrochloride (MEA) or mercaptoethanol in 50% DMSO/water is a good first choice. Make up about 10% of the column volume of this mixture; dissolve the thiolated compound in half the final volume of deionized water, then make up to final volume with DMSO. Note that mixing DMSO and water generates heat, so let the cleaning solution cool back down to room temperature before using. After injection, run the column as slowly as you can - the longer this solution remains in contact with the gold deposits, the more effective it will be. For maximum efficacy, set it up to run overnight. We suggest leaving the detector and recorder running (slowly!) to provide a record of when the various materials are eluted and to verify that the cleaning solution has cleared and absorption has returned to baseline.

• 1 M acetic acid in DMSO may be effective for colloidal gold, particularly the red-purple material that can remain behind after repeated Nanogold separations. To remove the more stubborn purple and blue colloidal gold deposits, use the thiol wash first.

• Cleaning procedures for many columns are available on manufacturer web sites; for example, GE (Amersham Pharmacia) and Bio-Rad provide comprehensive instructions for cleaning their gel filtration columns on the Amersham Pharmacia web site:

  Cleaning GE gel filtration columns
  Cleaning instructions for Bio-Rad Bioscale columns
  

  Generally, 1 M NaOH followed by water is recommended to remove proteins, and 70% ethanol, 70% formic acid or 70% acetonitrile to remove hydrophobic contaminants.

More information:

• Guide to Gold Cluster Labeling: Separating your conjugate
• Technical help: Nanogold labeling reagents
• Previous discussion: Labeling and separation strategies
• Guide to Gold Cluster Labeling: Overview

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Enzyme Metallography for Ultrasensitive Electrical Detection on DNA Chips

A chip design with 42 measuring spots was used for the electrical detection of DNA: the measuring spots are 10 micrometer wide microstructured electrode gaps prepared on a silicon oxide substrate covered by 5 nm of titanium and 100 nm of gold using standard photolithographic processes. The chip surface was modified with (3-glycidyloxypropyl) trimethoxysilane to immobilize a 30 base pair (bp) amino-modified oligonucleotides. Different capture DNA probes were then immobilized on the chip: a complementary probe (NS150), a probe with one mismatch (NS151), a probe with three mismatches (NS153), and a noncomplementary probe (N7), each 30 bp long. Two electrode pairs on each chip were left unmodified as controls for the background signal.

For hybridization of the biotin-modified target probes on the chips, the probes were dissolved in 5 x SSC + 0.1% SDS, then incubated with the chips for at least 1 hour in a humidity chamber, then washed for 5 minutes in 2 x SSC + 0.1% SDS, then washed for a further 5 minutes in 5 x SSC. After a final wash for 5 minutes in 0.2 x SSC, the chips were again dried under a nitrogen. Detection using enzyme metallography was compared with that for silver-enhanced gold. For the gold, 100 microliters of a 1:100 dilution of streptavidin-5nm gold nanoparticle in PBS pH 7.4 with 0.1% BSA was applied to each chip and incubated for 1 h at 37°C in a humidity chamber. The chips were then washed in PBS, pH 7.4, for 5 min, briefly rinsed with distilled water to remove any excess chloride ions, then silver enhanced with 300 microliters of British Biocell SEKL 15 reagent for 2 or 4 minutes. The chips were washed in distilled water to stop the reaction and dried under nitrogen. The chips were finally measured using a customized readout device; the reader was constructed around a special socket, which holds the chip during the measurement and contacts all 42 electrode gaps on our chip for a semiparallel measurement; it also includes a multiplexed ohm meter controlled by an embedded PC. The procedure was repeated until a significant drop in the measured resistance was detected.

Enzyme metallographic detection was accomplished using a streptavidin-peroxidase polymer. The substrates were incubated with a 1:1000 dilution of the streptavidin peroxidase polymer solution in PBS pH 7.4 with 0.05% Tween-20. A 100 microliter portion of this solution was applied per chip and incubated at 20°C for 1 hour. The chips were then washed 6 x 5 minutes in phosphate-buffered saline, pH 7.4, with 0.05% Tween-20 to remove any excess of bound enzyme complex, rinsed briefly with distilled water to remove any chloride ions, and dried under a stream of nitrogen. The chip was then developed with the enzyme metallographic reagent: after 5 minutes, development was stopped by washing the chips in water and then drying under nitrogen. The custom-built readout device was used to measure the resistance on the chip. A further enhancement was conducted (if necessary) using the British Biocell silver enhancement kit used for the gold nanoparticles.

Examination of the two chips using AFM revealed a significant reduction in background signal, with fewer silver deposits in the substrate areas adjacent to the spotted region; also, a highly defined separation was found between the spot region with deposited metal of about 130 nm height and the surrounding background, with no detectable metal clusters. Furthermore, the electrode regions that are situated inside the spot area also show a clearly suppressed metal deposition with the enzymatic process compared with images from the metal-catalyzed case. This lower background signal enables the enzyme-based detection scheme to be significantly more sensitive than the nanoparticle-based system: it was found to be possible to detect biotin-modified target DNA concentrations as low as 500 fM, corresponding to 50 amol of biotin-modified target DNA. A further increase in the sensitivity of the system seems feasible with further optimization of the development process. These results indicate that enzyme metallography can offer a significant improvement for electrical detection on biochips, bringing closer robust, portable, multiplexed biochip diagnostics.

Reference:

Moller, R.; Powell, R. D.; Hainfeld, J. F., and Fritzsche, W.: Enzymatic control of metal deposition as key step for a low-background electrical detection for DNA chips. Nano Lett., 5, 1475-1482 (2005).

• Abstract (courtesy of PubMed (Medline)).

More information:

• Enzyme Metallography for correlative labeling (our Microscopy & Microanalysis 2004 abstract)
• Enzyme Metallography - a New Staining Method (our Microscopy & Microanalysis 2002 abstract)
• Enzyme Metallography for in situ hybridization
• Other product and research applications

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Nanogold® Labeling for ICP-Mass Spectrometry Assays

The authors describe a novel application of ICP-MS-linked metal-tagged immunophenotyping. Expression of intracellular oncogenic kinase BCR/Abl, myeloid cell surface antigen CD33, human stem cell factor receptor c-Kit and integrin receptor VLA-4 were investigated using model human leukemia cell lines. Antigens to which specific, non cross-reactive antibodies are available which may be distinguishably tagged can be determined simultaneously, or multiplexed. Four commercially available lanthanide tags (Au, Sm, Eu, and Tb) conjugated to different secondary antibodies were shown to enable a 4-plex assay. Results obtained by ICP-MS were compared with data from flow cytometry.

ICP-MS as an analytical detector possesses several advantages that enhance the performance of immunoassays. It has high precision, low detection limits, and a large dynamic range, both for each antigen and between antigens. It shows lower matrix effects from other sample components (contaminating proteins in the sample have no effect on elemental analysis), and lower background from plastic containers and plates (plastic containers do not cause interference on elemental detection, as they can with fluorescence). Results are independent of non-specific background and analytical response from incubation or storage times, since protein degradation does not affect analysis of an elemental tag. The multiplexing potential is very large (potentially, up to 167 isotopes; realistically, around 100 distinguishable tags), and better spectral resolution (abundance sensitivity) is obtained. Because signals from elemental tags are essentially non-overlapping, there is no need to compensate for equal intensity for each application.

Lanthanide-labeled (Eu, Tb, Sm) affinity reagents were purchased from Perkin Elmer Life Sciences (Turku, Finland): Eu-N1-anti-rabbit (DELFIAR #AD0082), Tb-N1-Streptavidin (StrAv-Tb; DELFIAR #AD0047); Sm-N1-Streptavidin (StrAv-Sm; DELFIAR# AD0049). Human monocyte cell lines MBA-1 and MBA-4 were derived from Mo7e by retroviral induction of the p210 BCR/Abl cDNA; expression of the BCR/Abl oncogenic kinase confers growth factor independence. These cell lines serve as model systems for studying human megakaryocytic leukemia and drug inhibition of the BCR/Abl signal transduction pathways. Cells were propagated in alpha-MEM, supplemented with 10% FBS and 2 mM l-glutamine, in a humidified incubator at 37°C and 5% CO₂. Cells were split every 34 days and viability checked with trypan blue (90% viable). Antibodies used were anti-CD33 (mouse monoclonal), biotinylated anti-human SCFR/c-Kit antibody (CD117), biotinylated anti-human Integrin alpha4 (CD49d or VLA-4) mouse monoclonal antibody; anti-BCR antibody raised in rabbit (Cell Signaling Tech) which detects total levels of endogenous BCR and the p210 kDa BCR/Abl fusion protein. Anti-IgG1 kappa mouse, and anti-IgG1 rabbit (BD PharMingen) were used for negative controls. Antibodies were diluted in PBS/10% FBS.

Cells in logarithmic growth phase were collected by centrifugation (1200 rpm, 10 min), washed once in PBS/10% FBS and counted in a hemocytometer. The cells were then distributed into round-bottom 5 mL flow cytometry tubes in triplicates for staining. Cells were Incubated with primary antibodies on ice for 30 minutes, permeabilized for 30 minutes at room temperature, then washed and non-specific antigen sites were blocked with PBS/10% FBS for 15 minutes at room temperature; cells were then stained for intracellular antigens. After secondary antibody incubation (30 minutes on ice), cells for ICP-MS analysis were washed twice, centrifuged and pellets were dissolved in concentrated HCl (75 microliters per tube) for 10 minutes at room temperature. Nanogold anti-mouse-IgG was prepared in three different dilutions (1:10, 1:25, and 1:50), and added to the washed cell pellets for another 30 minutes on ice. All cells were washed twice with PBS/FBS and left as a pellet after the second wash. Pellets were then frozen overnight and dissolved in 34% HCl next day and run on ICP-MS. As an internal standard for ICP-MS measurements, 75 microliters of 1 ppb Ir (Iridium) solution was added to each tube. Experimental measurements were made on a commercial ICP-MS instrument ELAN DRCPlusk (Perkin Elmer SCIEX).

An immunophenotyping experiment was set up to simultaneously identify two cell membrane antigens (CD33 and c-Kit) and the intracellular proteins BCR and p210BCR/Abl. Cells were processed as described above, permeabilized for 30 minutes, blocked 15 minutes with PBS/10% FBS, then incubated with a mix of primary antibodies (anti-CD33, biotinylated-anti-c-Kit and anti-BCR) for 30 minutes at room temperature. After one wash with PBS/10% FBS, metal-conjugated reagents (Nanogold anti-mouse, streptavidin-Tb, and anti-rabbit-Eu) were applied to the cells. Comparable signals were obtained whether the secondary tags were added all at once, or separately, indicating that there is no interference from each metal-tag when used in a multiplex assay. Finally, for comparison with flow cytometry, a multiplex four-antigen identification by ICP-MS and was conducted. Live cells were stained with anti-CD33 and anti-c-Kit antibodies, permeabilized; stained with a mix of anti-BCR and Nanogold anti-mouse, and followed by streptavidin-Sm and anti-rabbit-Eu. The final incubation included anti-VLA-4, followed by StrAv-Tb for ICP-MS.

The results of these experiments provide proof-of-principle that ICP-MS can be used in the multiplexed molecular analysis of human leukemia cell lines. Both cell surface and intracellular proteins were clearly detected, and it was clearly established that four proteins could be quantitatively analyzed, proving the multiplex potential of this approach. Although multiplexing using metal-conjugated reagents is in a very early stage of research and feasibility studies, it is already apparent that more than four antigens could be accurately detected simultaneously using the ICP-MS instrument.

Reference:

Ornatsky, O.; Baranov, V. I.; Bandura, D. R.; Tanner, S. D., and Dick, J.: Multiple cellular antigen detection by ICP-MS. J. Immunol. Methods., 308, 68-76. (2006).

• Abstract (courtesy of Science Direct).
• Reprint (PDF) (courtesy of Science Direct).

More information:

• Nanogold conjugates: Catalog information
• Nanogold-antibody conjugates: Product information
• Technical help: Silver enhancement
• Technical help: Nanogold conjugates
• References: Nanogold conjugates

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Other Recent Publications

Reference:

Bukreyev, A.; Yang L.; Zaki S. R.; Shieh W. J.; Rollin P. E.; Murphy B. R.; Collins P. L., and Sanchez A.: A Single Intranasal Inoculation with a Paramyxovirus-Vectored Vaccine Protects Guinea Pigs against a Lethal-Dose Ebola Virus Challenge. J. Virol., 80, 2267-2279 (2006).

• Abstract (courtesy of the Journal of Virology).

Want nanoparticles of cobalt rather than gold? Check the method of Salgueiriño-Maceira and group in their recent Langmuir article. Low size polydisperse cobalt spheres with an average diameter of 95 nm were synthesized by using a borohydride reduction method: 0.1 mL (0.4 M, 4 x 10^-5 mol) of aqueous cobalt chloride hexahydrate was added to an aqueous solution (100 mL) of sodium borohydride (4.4 mM, 4.4 x 10^-4 mol) and citric acid monohydrate (2 x 10^-6 M, 2 x 10^-7 mol) under mechanical stirring. Immediately following the cobalt reduction, the particles were coated with a thin layer of silica using 400 mL of an ethanolic solution containing 15 microliters of tetraethoxysilane (98%) was added. After 15 minutes, the solution was centrifuged, and the precipitate was redispersed in ethanol (40 mL). The large uniform cobalt spheres were found to consist of smaller metallic cobalt clusters, explaining their superparamagnetic behavior.

Reference:

Salgueirino-Maceira, V.; Correa-Duarte, M. A.; Farle, M.; Lopez-Quintela, M. A.; Sieradzki, K., and Diaz, R.: Synthesis and Characterization of Large Colloidal Cobalt Particles. Langmuir, 22, 1455-1458 (2006).

• Abstract (courtesy of PubMed (Medline)).

Krichevsky and colleagues contribute a new method for preparing silver-gold nanowires in their recent article in Langmuir article. Thin, long gold/silver nanowires were grown on substrates in thin surfactant solution films; the growth process occurred exclusively in thinning aqueous films as the water evaporated, forming elongated surfactant template structures. The nanowire growth solution was prepared by combining Au(I) and Ag(I) solutions with Au:Ag molar ratio 1:1, in the presence of a high surfactant concentration, reducing agent (ascorbic acid), and sodium hydroxide addition. The standard growth solution contained 10 mL of 0.05M cetyltrimethylammonium bromide (CTAB) with 2.5 micromol of HAuCl₄ (0.5 mL of 5 mM solution), 2.5 micromol of AgNO₃ (125 microliters of 20 mM solution), and 55 micromol ascorbic acid. To this solution was added 40 microliters of 1 M NaOH (40 micromol) while stirring, raising the pH of the solution to a value of ~5 and initiating the growth process. Modifications of this process were also tested: addition of 25 microliters of 3-4 nm gold seed nanocrystal solution after base addition; variation of the Ag:Au mole ratio to the extremes where no gold or no silver was present; and leaving out OH^- addition. Nanowire growth required the presence of a relatively high concentration of silver ions (typical Ag:Au mole ratio of 1:1), and tuning the pH value to about 5 in the growth solution. EDS analysis indicated nanowire composition of 85-90% gold and 15-10% silver.

Reference:

Krichevski, O.; Tirosh, E., and Markovich, G.: Formation of gold-silver nanowires in thin surfactant solution films. Langmuir, 22, 867-870 (2006).

• Abstract (courtesy of PubMed (Medline)).

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