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N A N O P R O B E S     E - N E W S

Vol. 6, No. 6          June 9, 2005


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Nanogold® Conjugates
Buy nowCovalently bonded to antibody, IgG or streptavidin

Nanogold®-Streptavidin as a Tertiary Probe for Pre-embedding Labeling

There are several potential advantages to using a labeled tertiary probe for immunolabeling:
  • Specificity is often higher, and may be increased by selecting a probe against a hapten or antigen which is not present endogenously, or which may be more effectively blocked.
  • Sensitivity can be increased because several molecules of labeled detection reagent may bind to the appropriate haptenated secondary probe.
  • When a labeled secondary antibody is not available or is difficult to prepare, a labeled tertiary can provide the simplest method for effective detection.

Minaki and co-workers provided a good example of the utility of tertiary labeling in their recent report in Neuroscience Research, using biotinylated secondary antibody and Nanogold-streptavidin to localize a monoclonal hamster primary antibody and label components of developing neurons.

Postmitotic neural precursors are generated in the ventricular zone (VZ) of the developing neural tube, and immediately migrate to the mantle layer (ML), where they differentiate into mature neurons. The regulation of neuronal differentiation and migration has been studied extensively, but the behavior of the early postmitotic precursors migrating toward the ML is largely unknown. Using PCR to examine its distribution in different mouse tissues, the authors identified Neph3, an immunoglobulin domain-containing transmembrane protein, as a specific marker for early postmitotic neural precursors in the VZ of the developing spinal cord. The distribution of Neph3 was then investigated using immunofluorescence and immunoelectron microscopy. Immunofluorescence revealed that early neural precursors in the VZ possessed not only piaconnected processes but also ones that reached the ventricle.

This apical extension of processes was confirmed by analyzing another early postmitotic marker, Dll1 mRNA, which was actively transported toward the ventricle and accumulated at the termini of the processes. Double staining with an anti-ZO1 antibody indicated that Dll1 mRNA-containing processes reached the apical plane, at which adherens junctions (AJs) were formed around the apical end of processes extending from Neph3- and Dll1 mRNA-positive postmitotic precursors.

Tissue sections were prepared using a cryostat (12 micrometer thickness). The sections were incubated with Hamster anti-Neph3 monoclonal antibody raised against the extracellular domain of Neph3, followed by incubation with a biotin-conjugated secondary antibody; the sections were then incubated with Nanogold-streptavidin, and fixed with 1% glutaraldehyde. The antigen-bound gold particles were silver enhanced using HQ Silver at 25°C for 8 min, then postfixed with 1% osmium oxide in 0.1 M PB at 4°C for 1 h. The sections were stained with uranyl acetate in a moist chamber at 4°C for 30 min, dehydrated through ethanol and propylene oxide, then embedded in epoxy resin. Stained sections were further sectioned to 5070 nm and observed in the electron microscope. Immunoelectron microscopic analysis confirmed the idea that postmitotic precursors formed AJs: analysis of the staining showed Neph3 signals at the cellcell contact sites of the processes near the ventricle; some of the signals are detected on the AJs formed between the processes.

An in vitro model system - the subcellular localization of Neph3 protein in E-cadherin-expressing L fibroblasts (EL cells) - was used to investigate which cell types expressed Neph3, and its role adhesion. Neph3 protein expressed in EL cells was localized at cellcell contact sites; colocalization of Neph3 with E-cadherin indicated that Neph3 protein was localized at AJs in EL cells. Furthermore, Neph3 protein was localized at AJs formed between two Neph3-expressing cells, suggesting that localization of Neph3 protein at AJs is regulated by a homophilic trans-interaction; this was confirmed using a cell aggregation assay. Parental B300.19 cells did not form aggregates, but when C-terminally EGFP-tagged Neph3 protein was expressed in B300.19 cells, they aggregated efficiently, clearly indicating that Neph3 protein has cellcell adhesion activity. To determine whether the aggregation was mediated by homophilic interactions of Neph3 protein or heterophilic interactions between Neph3 protein and other cell adhesion molecule(s), Neph3-EGFP-expressing B300.19 cells were cocultured with parental B300.19 cells labeled with DiI; the parental B300.19 cells did not coaggregate with Neph3-EGFP-expressing cells, indicating that the aggregation of Neph3 is mediated by homophilic interactions.

In summary, these observations suggest that migrating early postmitotic neural precursors in the VZ of the developing spinal cord form a neuroepithelial cell-like bipolar morphology. These communicate with neighboring cells through AJs.

Coincidentally, Tarabal and colleagues describe a similar tertiary labeling method in a recent Molecular and Cellular Neuroscience paper on protein retention and blockade of cell death in motor neurons. A pre-embedding immunolabeling procedure was used to detect PDI (protein disulfide isomerase) and HERC1 (a giant protein that stimulates guanine nucleotide release on some GTPases) immunoreactivity in lumbar spinal cords from E10 or E16 chick embryos. Spinal cords were dissected and fixed with 3% paraformaldehyde, 0.1% glutaraldehyde and 15% picric acid in 0.1 M phosphate buffer for 5 h. 50-micron transverse sections obtained with a vibratome were treated with 50 mM glycine for 30 minutes, then with 10% normal goat serum in 0.01% saponin in phosphate-buffered saline. Sections were then incubated overnight at 4°C with either mouse anti-PDI (1/300) or rabbit anti-HERC1 (#407, 1/1000) primary antibodies. After washing, they were incubated for 1 hour with the appropriate biotinylated anti-mouse or anti-rabbit secondary antibodies (1/100), then processed either according to ABC peroxidase protocol (Vector Laboratories), or incubated with Nanogold-streptavidin (1/100) for 1 hour followed by silver enhancement. Labeled sections were flat embedded in Embed 812, sectioned, counterstained with uranyl acetate and lead citrate, and observed in the electron microscope.

 

References:

  • Minaki, Y.; Mizuhara, E.; Morimoto, K.; Nakatani, T.; Sakamoto, Y.; Inoue, Y.; Satoh, K.; Imai, T.; Takai, Y., and Ono, Y.: Migrating postmitotic neural precursor cells in the ventricular zone extend apical processes and form adherens junctions near the ventricle in the developing spinal cord. Neurosci. Res., 52, 250-262 (2005).
  • Tarabal, O.; Caldero, J.; Casas, C.; Oppenheim, R. W., and Esquerda, J. E.: Protein retention in the endoplasmic reticulum, blockade of programmed cell death and autophagy selectively occur in spinal cord motoneurons after glutamate receptor-mediated injury. Mol. Cell. Neurosci., 29, 283-298 (2005).

 

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Nanogold Labeling: Aggregation, Antibodies and Oligonucleotides

More from our technical help files...

How do I prevent aggregation when I use Nanogold® labeling reagents?

Our instructions for Monomaleimido Nanogold and Mono-Sulfo-NHS-Nanogold advise dissolving the reagent in a small amount of DMSO (dimethylsulfoxide), then diluting with water or aqueous buffer. When you add the DMSO, you may notice some residual solid: this is normal, and is not undissolved Nanogold, but the buffer salt with which it was lyophilized. Once the water is added this will dissolve and ensure that the Nanogold solution is at the optimum pH for labeling. To ensure that the Nanogold is completely dissolved, vortex this solution before use.

However, during Nanogold® labeling, you may notice the formation or deposition of some solid, indicating aggregation or precipitation of the reagent or conjugate, especially with smaller molecules such as peptides. If you notice this, or find that a significant proportion of the reagent precipitates and does not enter chromatography media for separation, we suggest the following:

  • Use the maximum proportion of DMSO that you can in the reaction mixture. You can use up to 20% without harm to most proteins. Nanogold is highly soluble in DMSO, and this will help to ensure that Nanogold and conjugates remain solvated.
  • Vortex before injection onto columns for separation, especially if you have used a membrane centrifugation filter or other concentrating device: these can induce precipitation. If this has occurred, adding a few drops of DMSO may help redissolve any precipitate.
  • Use of proper stoichiometry can help avoid aggregation. If you are labeling a molecule that is larger than Nanogold (MW about 15,000 or more), use a 3-fold to 5-fold excess of Nanogold reagent, especially for Monomaleimido Nanogold and NTA-Ni(II)-Nanogold.
  • Consider using an alternative reagent, particularly if you find aggregation when you are using Monomaleimido Nanogold to label a thiol-modified biomolecule. Because of the natural affinity of thiols for gold, interaction of excess thiols with the gold particles can cause aggregation; in this cause, if you can label at an amine site instead, try Mono-Sulfo-NHS-Nanogold.

Which Nanogold labeling reagent should I use to label antibodies - IgG, Fab' or Fab?

Generally, we recommend Monomaleimido Nanogold for antibody labeling. Because labeling with this reagent is directed to the hinge region in IgG molecules or Fab' fragments, it does not hinder the antigen combining region from binding to its target. However, excellent labeling may also be obtained by using Mono-Sulfo-NHS-Nanogold, and in tests we have found that antibodies labeled in this manner can produce labeling equivalent to or even better than those labeled with Monomaleimido Nanogold.

You might consider labeling with Mono-Sulfo-NHS-Nanogold in the following situations:

  • You cannot readily isolate the reduced antibody, or selectively reduce the hinge thiol, or you are working with a different type of immunoglobulin such as IgA or IgY.
  • You need to label non-Fc-containing fragments, but your antibody is of a class that cannot readily be digested using pepsin or ficin to yield F(ab')2. In this case, use papain digestion to generate Fab fragments and label with Mono-Sulfo-NHS-Nanogold.
  • Labeling with Monomaleimido Nanogold results in aggregation. In some cases, the affinity of thiols for gold even in the absence of maleimides causes aggregation, particularly in situations where the antibody is difficult to quantitate and the ratio of Nanogold to antibody may be less than ideal. In this case, using Mono-Sulfo-NHS-Nanogold does not require the thiol, and one of the mechanisms of aggregation is removed.

I'm labeling an oligonucleotide, so I need to calculate labeling using the extinction coefficient at 260 nm rather than 280 nm. Can you give a value for this?

The spectrum of Nanogold is shown below from 260 nm to 820 nm:

[Nanogold UV/vis Spectrum (6k)]
UV/visible spectrum of unconjugated Nanogold, recorded from 260 nm to 820 nm, with absorbance values at 5 wavelengths and extinction coefficients at 260 nm, 280 nm and 420 nm.

Given that the extinction coefficient at 280 nm is 3.0 x 105 M-1cm-1, the extinction coefficient at 260 nm therefore calculates to be 4.3 x 105 M-1cm-1.

For undecagold, the spectrum is shown below:

[Undecagold UV/vis Spectrum (6k)]
UV/visible spectrum of unconjugated undecagold, recorded from 260 nm to 820 nm, with absorbance values at 5 wavelengths and extinction coefficients at 260 nm, 280 nm and 420 nm.

Given that the extinction coefficient at 280 nm is 16.8 x 104 M-1cm-1, the extinction coefficient at 260 nm therefore calculates to be 24.1 x 104 M-1cm-1.

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Nanogold®-Fab' Labeling in Platelets: Regulation of Granule Secretion

Nanogold®-Fab' is the smallest commercially available immunogold reagent capable of visualization in a regular transmission electron microscope; a comparison of the size of Nanogold-Fab' with a conventional 5 nm colloidal gold-IgG conjugate is shown below.

[Size and resolution comparison: colloidal gold-IgG vs. Nanogold-Fab' (21k)] 
Size comparison of Nanogold-Fab' with conventional 5 nm colloidal gold-IgG probe, showing overall probe size and distance of gold from target.

This small size gives Nanogold-Fab' several advantages over colloidal gold probes:

  • Better penetration, up to 40 microns into cells and tissue sections.
  • Higher density of labeling.
  • Greater access to hindered or restricted antigens.

These advantages were demonstrated in a recent article in Blood by Flaumenhaft, Dvorak and group, who used it in their studies on evaluate the role of the actin cytoskeleton in platelet granule secretion. Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. This has led to the supposition that cytoskeletal contraction promotes the interaction of granules with one another and with membranes of the open canalicular system, and hence facilitates granule release; yet the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. The authors therefore investigated the effects of latrunculin A and cytochalasin E on granule secretion. Low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced alpha-granule secretion, with comparatively modest effects on dense granule secretion. However, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced alpha-granule secretion, but stimulated dense granule secretion.

Stimulation of platelets for 10 minutes with the thrombin receptor agonist peptide, SFLLRN, results in dramatic platelet shape change with centralization and loss of alpha-granules; in contrast, stimulation of platelets with the phorbol ester PMA for 10 minutes results in only modest shape change. To determine whether or not P-selectin remains associated with alpha-granules, platelets were labeled using an antibody directed at the cytoplasmic tail of P-selectin. With PMA, these studies demonstrated that P-selectin did not migrate to the outer surface of the plasma membrane within this time frame but remained largely associated with alpha-granule membranes; however, immunogold staining for P-selectin in platelets exposed to SFLLRN demonstrated substantial surface expression of P-selectin.

For immunogold staining, purified human platelets were fixed in 4% paraformaldehyde in 0.02 M PBS, pH 7.4, for 1 hour at room temperature, then washed in 0.02 M PBS, pH 7.4, transferred to microtubes and centrifuged at 1500 g for 1 minute. They were then resuspended in molten 2% agar and quickly recentrifuged. The resulting agar pellets containing the platelets were washed in PBS, immersed in 30% sucrose in 0.02 M PBS, pH 7.4 overnight at 4°C, embedded in OCT compound, and stored in -176°C liquid nitrogen for subsequent use. Frozen 10-micron sections were cut with a standard cryostat, collected on precleaned glass slides, and air dried for 20 minutes before staining.

Immunogold staining and processing were performed at room temperature on cryostat sections mounted on glass slides, as follows:

  1. Wash in 0.02 M PBS, pH 7.4, for 5 minutes.
  2. Immerse in 50 mM glycine in 0.02 M PBS, pH 7.4, for 10 minutes.
  3. Wash in 0.02 M PBS, pH 7.4, for 5 minutes.
  4. Immerse in 5% normal goat serum for 20 minutes.
  5. Incubate in the primary antibody, polyclonal rabbit antibody directed against the cytoplasmic tail of P-selectin, at a dilution of 1:50 in 0.02 M PBS for 60 minutes.
  6. Wash 3 x 5 minutes in 0.02 M PBS, pH 7.4.
  7. Incubate in the secondary antibody (Nanogold-Fab goat antirabbit IgG) at 1:50-100 dilution in 0.02 MPBS, pH 7.4, for 60 minutes.
  8. Wash 3 x 5 minutes in 0.02 M PBS, pH 7.4.
  9. Postfix in 1% glutaraldehyde in 0.02 M PBS, pH 7.4, 2 minutes.
  10. Wash 3 x 5 minutes in distilled water.
  11. Develop with HQ Silver enhancement solution for 6 to 10 minutes in the darkroom.
  12. Wash 2 x 2 minutes in distilled water.
  13. Immerse in 5% sodium thiosulfate for 1 minute.
  14. Wash 3 x 5 minutes in distilled water.
  15. Fix in 1% osmium tetroxide in Sym-Collidine buffer, pH 7.4 for 10 minutes.
  16. Wash in 0.05 M sodium maleate buffer, pH 5.2, for 5 minutes.
  17. Stain with 2% uranyl acetate in 0.05 M sodium maleate buffer, pH 6.0, for 5 minutes.
  18. Wash in distilled water for 5 minutes.
  19. Dehydrate in graded ethanols, then infiltrate with a propylene oxide-eponate sequence.
  20. Embed by the inversion of eponate-filled plastic capsules over the slide-attached tissue sections.
  21. Polymerize at 60°C for 16 hours.
  22. Separate the eponate blocks from glass slides by brief immersion in liquid nitrogen.
  23. Cut thin sections with a diamond knife with an ultratome. Collect sections on uncoated 200-mesh copper grids (Ted Pella).
  24. View unstained grids in the transmission electron microscope.

Incubation of permeabilized platelets with low concentrations of latrunculin A-primed platelets for Ca2+ or guanosine triphosphate (GTP)-gamma-S induced alpha-granule secretion. Latrunculin A-dependent alpha-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates alpha-granule and dense granule secretion.

 

Reference:

Flaumenhaft, R.; Dilks, J. R.; Rozenvayn, N.; Monahan-Earley, R. A.; Feng, D., and Dvorak, A. M.: The actin cytoskeleton differentially regulates platelet alpha-granule and dense-granule secretion. Blood, 105, 3879-3887 (2005).

 

Reference for full protocol:

Feng, D.; Crane, K.; Rozenvayn, N.; Dvorak, A. M., and Flaumenhaft R.: Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2. Blood, 99, 4006-4014 (2002).

 

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Nanowires from Gold Enhanced Nanogold®

Development of preprogrammable conductive nanowires is a requisite for the fabrication of nanoscale electronics based on molecular assembly. Nanogold® is well suited to this purpose since it may be linked using a variety of processes to a range of substrates which may then be assembled or incorporated into nanowires or other structures. In their recent paper in the Journal of the American Chemical Society, Nishinaka and group describe the synthesis of conductive metal nanowires from nucleoprotein filaments, complexes of single- or double-stranded DNA, and RecA protein.

A genetically engineered RecA derivative, 353C, possessing a reactive, surface-accessible cysteine residue was reacted with Monomaleimido Nanogold particles. Filament formation reactions were carried out at 37°C for 20 minutes, contained in 50 microliters of 20 mM Tris-acetate (pH 7.5), 1 mM magnesium acetate, 2 mM ATP-gamma-S, 10 x 10-6 M lambda DNA, and 3.3 x 10-6 M 353C RecA (concentration of DNA expressed as moles of nucleotide residues). Monomaleimido Nanogold was added to a reaction mixture to a final concentration of 60 x 10-6 M. After incubation for 5 minutes at room temperature, the reaction mixture was immediately loaded onto a Sepharose 2B gel filtration column equilibrated with 20 mM HEPES (pH 7.5), 1 mM magnesium acetate, and 1 mM ATP-gamma-S. When fixation of the RecA filament was needed, the column fraction containing RecA filament was reacted with 0.5% glutaraldehyde for 8 minutes and the reaction was stopped by 0.1 M glycine. This procedure yielded nucleoprotein filaments with Nanogold particles attached.

The template-based gold particles were then enlarged by either silver enhancement using LI Silver, or gold enhancement using GoldEnhance EM. A droplet of sample solution containing the Nanogold-labeled 353C RecA filaments was placed onto a chip of silicon wafer (for SEM) or a carbon-coated copper grid (for TEM). The solution was incubated for 5-15 seconds and wicked with filter paper. These samples were immediately gold enhanced using the method of Keren or with GoldEnhance EM; for silver enhancement, LI Silver solution was used. Exposure times varied from 30 s in the TEM experiments to 3-30 min for the SEM experiments. Usually, continuous nanowires about 200 nm in diameter were obtained after 4 min of gold enhancement using the method of Keren. To stop the reaction, the sample was dipped into 200 mL of water 1-3 times, in 50 mL of acetone (if necessary), and then dried at room temperature. SEM images were obtained using an accelerating voltage of 10 kV and emission current of 10 microamps; TEM images were obtained at an accelerating voltage of 100 kV. For AFM observation, a droplet of sample solution was placed onto (aminopropyl) triethoxysilane(AP)-mica (positively charged mica functionalized with (aminopropyl)triethoxysilane). The solution was incubated for 5-15 seconds then wicked with filter paper. AFM images were acquired with a NanoScope IV MultiMode system (Veeco Instruments Inc., Santa Barbara) in tapping mode.

Conductivity measurements using a four-point probe four-point probe method. Silicon chips fabricating 80 lithographically patterned electrodes with gold-stripes 30 microns wide by 400 microns long were prepared over a silicon nitride membrane. Each electrode was separated by a 2 micron gap, although four were directly connected without any gap as a reference. Nanogold-labeled 353C RecA filaments were spread randomly over the patterned electrodes, then gold enhanced to form metal nanowires. A nanovoltmeter and a picoammeter were connected to the electrodes through four probe needles, and the I-V curve of the circuit was measured in the 0.2 - 2 x 10-6V range. The I-V curve was linear with small deviations, indicating that the observed nanowire showed ohmic behavior (R = 8.9 ohms for a single nanowire with a diameter of approximately 200 nm).

This approach shows that programming information can be encoded in DNA sequences so that an intricate electrical circuit can be constructed through self-assembly of each component. As the RecA filament has higher degree of stiffness than double-stranded DNA, it provides a robust scaffold that allows the fabrication of more reliable and well-organized electrical circuitry at the nanoscale. Furthermore, the homologous pairing provides sequence-specific junction formation as well as sequence-specific patterning metallization.

 

Reference:

Nishinaka, T.; Takano, A.; Doi, Y.; Hashimoto, M.; Nakamura, A.; Matsushita, Y.; Kumaki, J, Yashima, E.: Conductive Metal Nanowires Templated by the Nucleoprotein Filaments, Complex of DNA and RecA Protein. J. Amer. Chem. Soc., 127, 8120-8125 (2005).

 

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Other Recent Publications

Hot on the heels of last month's report featuring NTA-Ni(II)-Nanogold®, Collins and co-workers report the use of the same reagent for the characterization of cell surface pili. Type IV pili are cell surface organelles found on many Gram-negative bacteria which mediate functions including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits, capable of rapid polymerization or depolymerization that generates large motor forces. A specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber was detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, with purified PilQ oligomer added, showed that PilQ was uniquely located at one end of the pilus fiber, forming a "mallet-type" structure. PilQ localization was confirmed by the incorporation of His-tagged PilQ into oligomerized PilQ, and detection with NTA-Ni(II)-Nanogold. The gold particles were effectively imaged in preparations negatively stained with ammonium molybdate/trehalose to ensure that the gold particles could be readily visualized. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-Å resolution and comparison with a previously determined structure of PilQ at 12-Å resolution indicated that binding of the pilus fiber induced dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. These results demonstrate that the PilQ structure exhibits a dynamic response to the binding of transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion.

Reference:

Collins, R. F.; Frye, S. A.; Balasingham, S.; Ford, R. C.; Tonjum, T., and Derrick, J. P.: Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ. J. Biol. Chem., 280, 18923-18930 (2005).

El-Sayed and colleagues report the use unique surface plasmon resonance (SPR) signatures of gold nanoparticles conjugated to colloidal gold nanoparticles and from gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies as biosensors for imaging in cell cultures with a nonmalignant epithelial cell line (HaCaT) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3) in the current Nano Letters, demonstrating that such sensors can function in living cells. Colloidal gold particles with average diameter 35 nm, prepared by citrate reduction of tetrachloroaurate, were adsorbed to the anti-EGFR antibody using conventional procedures. The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type cells with 600% greater affinity than to the noncancerous cells. This specific and homogeneous binding is found to give a relatively sharper SPR absorption band with a red shifted maximum compared to that observed when added to the noncancerous cells.

Reference:

El-Sayed, I. H.; Huang, X., and El-Sayed, M. A.: Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer. Nano Lett., 5, 829-834 (2005).

Supramolecular or macromolecular assembly is one of the primary applications of functionalized gold nanoparticles such as Nanogold®, and we have focused on these applications in previous articles. Deng and co-workers provide a good example in their recent paper in Angewandte Chemie, International Edition in English. They utilized a basic approach of hybridizing mono-DNA-functionalized gold nanoparticles with template-DNA strands to form the desired structures. Rolling-circle amplification was used to construct long, repetitive strands of DNA; in this method, a DNA polymerase uses a short (less than 100 bases long), circularized, single DNA strand as a template to synthesize long (more than 10,000 bases), linear, tandemly repetitive single DNA strands under isothermal conditions. 5 nm colloidal gold particles were stabilized by complexation with bis (para-sulfonatophenyl) phenylphosphine, precipitated with solid sodium chloride, and after centrifugation and careful supernatent removal, redispersed in deionized water. The shorter oligonucleotide-gold conjugate was prepared by mixing a 53-base thiolated oligonucleotide (DNA1) and phosphine-protected gold particles; the product, containing a mixture of gold particles conjugated with different numbers of DNA1, was separated by agarose gel electrophoresis. Gold nanoparticle assemblies were then prepared by mixing purified AuDNA1 (0.3-0.8 mM based on absorbance at 520 nm) with DNA templates at molar ratios (AuDNA1 : repeat of the template) of 1 : 1, 1 : 0.5, and 1 : 0.25 and annealing from 95°C to 4°C. Products were separated by electrophoresis on 0.5% agarose gel. Transmission electron microscopy of smaller assemblies containing from 3 to 6 repeats demonstrated the formation of complexes containing the required numbers of nanoparticles, and the mean separation for linear pairs of 18.5 ± 6.9 nm fitted well with the distance of 18.0 nm for a 53-base DNA duplex. Arrays formed with the longer DNA templates prepared by rolling circle amplification showed linearly arranged gold particles with separations consistent with the predicted structure.

Reference:

Deng, Z.; Tian, Y.; Lee, S.-H.; Ribbe, A. E., and Mao, C.: DNA-Encoded Self-Assembly of Gold Nanoparticles into One-Dimensional Arrays. Angew. Chem., Int. Ed. Eng., 44, 3582-3585 (2005).

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