Updated: August 13, 2003

N A N O P R O B E S     E - N E W S

Vol. 4, No. 8          August 13, 2003

This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.

Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing tech@nanoprobes.com.

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New Nanogold® Pre-Embedding Labeling Procedure

Microscopy & Microanalysis '03 included a new procedure for effective, reliable pre-embedding labeling with Nanogold® for electron microscopy. Xinran Liu and co-workers used the following procedure to localize the key synaptic protein, SynCAM (SynaptiC Adhesion Molecule), and also to identify synapses transfected with fluorescent fusion proteins of mutant or normal synaptotagmin 1 and 7. Hippocampal cultures were prepared from 1-2 day old Sprague-Dawley and transfected after 6 days using calcium phosphate transfection protocol with PCMV5-Syt1-EYFP and PCMV5-Syt7-EYFP transfection vectors; Cells were prepared for EM at days 10-11, 4-5 days after transfection. For endogenous protein localization, cells were fixed and processed after 14 divisions of primary culture. Immunogold labeling was conducted as follows:
  1. Fix cells in 0.1% glutaraldehyde, 3% paraformaldehyde in phosphate buffer (pH 7.4) for one hour at room temperature.
  2. Block with 2% normal goat serum, 1% bovine serum albumin, and 0.1 % saponin in the same buffer for 30 minutes.
  3. Incubate with primary antibody. In this case incubation was conducted for 1 hour at room temperature with either affinity-purified anti-SynCAM or a polyclonal antibody against GFP diluted 1 : 500.
  4. Wash in PBS, then incubate 1 hour with secondary Nanogold®-Fab' anti-rabbit IgG diluted 1 : 100 at room temperature.
  5. Silver enhance (HQ Silver, 8 minutes).
  6. Dehydrate the stained cells through a graded series of ethanol solutions to 100% and embed in PolyBed 812 epoxy resin.
  7. Stain ultrathin (70 nm) sections with 5% uranyl acetate.

Reference:

Liu, X.; Han, W.; Biederer, T.; Kavalali, E., and Südhof, T.: Identification of Endogenous/transfected Synaptic Proteins in Primary Neuronal Culture by a High-yield Immunogold Labeling. Microsc. Microanal., 9, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2003); Piston, D.; Bruley, J.; Anderson, I. M.; Kotula, P.; Solorzano, G.; Lockley, A., and McKernan, S. (Eds); Cambridge University Press, New York, NY, p. CD1498 (2003).

Two other reliable, effective methods for pre-embedding labeling are described in an application note on our web site. Grondin and Beaudoin report a procedure utilizing Nanogold with gold enhancement that produces high labeling with excellent ultrastructural preservation, while Dr. Susan Cheng and co-workers at NINDS have refined their pre-embedding procedure for the most uniform, consistent and reliable pre-embedding labeling, using Nanogold®-Fab' and HQ Silver.

References:

More information:

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Building Blocks for Nanotechnology: Chaperoned Nanogold®

Gold nanoparticles in the nanometer size range are important components for nanotechnology and nanobiotechnology. Over this size range, as their nature transitions from molecules to bulk metal, they demonstrate a variety of useful optical, chemical and electronic properties. Molecular control over the formation of the link between the biological molecule and metal particle enables the selective attachment of metal nanoparticles at sites where the metal particles impart useful functionality to the construct, or the templated assembly of supramolecular arrays of nanoparticles with novel properties.

Recently, McMillan and co-workers added to this growing field with their report of the fabrication of nanoscale two-dimensional arrays of quantum dots by the attachment of gold and semiconductor nanoparticles to crystalline arrays of hollow, double-ring protein structures. The structures were prepared by the modification of one of the three subunit proteins of the heat shock protein HSP60 from Sulfolobus Shibatae, a bacterium that lives in geothermal hot springs at temperatures up to 85°C and pH2; since this protein tolerates such conditions, it is easily separated and purified. Two classes of variants were made: in the first, a cysteine was engineered into a 28 amino acid loop that protrudes into the central cavity, giving a pore 3 nm in diameter. In the second, the loop was removed and the cysteine engineered into the apical domain itself to give an exposed, 9 nm cavity. Two-dimensional crystals of the variants were treated with 5, 10 or 15 nm colloidal gold stabilized with bis(p-sulfonatophenyl)-phenyl-phosphine; the 5 and 10 nm gold bound to the apical thiols of the variants with 3nm and 9nm pores respectively to give ordered arrays of gold particles, postulated to rest on top of the assembled protein complex, presumably tethered by the thiols, rather than inside the hollow core. Similarly, 4.5 nm CdSe/ZnS fluorescent quantum dots bound to the 3 nm pore variants showed similarly ordered regions. Cysteine-modified, loopless subunits were then labeled using Monomaleimido Nanogold® and reassembled; multiple Nanogold particles were observed on the inner surface of the structure, and XEDS measurements suggested that each pore contained up to nine Nanogold particles.

References:

  • Mogul, R.; McMillan, R. A.; Paavola, C. D.; Trent, J. D., and Zaluzec, N. J.: Self-Assembled 2D Protein Crystals as Templates for Ordered Metallic Nano-Arrays. Microsc. Microanal., 9, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2003); Piston, D.; Bruley, J.; Anderson, I. M.; Kotula, P.; Solorzano, G.; Lockley, A., and McKernan, S. (Eds); Cambridge University Press, New York, NY, p. CD274 (2003).

  • McMillan, R. A.; Paavola, C. D.; Howard, J.; Chan, S. L.; Zaluzec, N. J., and Trent, J. D.: Ordered nanoparticle arrays formed on engineered chaperonin protein templates. Nat. Mater., 1, 247-52 (2002).

These results demonstrate the range of options available for the biologically templated assembly of metal particles. Other biomolecules have also been used to template the assembly of Nanogold particles into supramolecular arrays, including self-assembling DNA crystals labeled site-specifically with Nanogold (Kiehl), and Nanogold-labeled lipids which can form gold-decorated liposomes (Hainfeld).

References:

  • Xiao, S.; Liu, F.; Rosen, A. E.; Hainfeld, J. F.; Seeman, N. C.; Musier-Forsyth, K., and Kiehl, R. A.: Selfassembly of metallic nanoparticle arrays by DNA scaffolding. J. Nanoparticle Res., 4, 313-17 (2002).

  • Hainfeld, J. F.; Furuya, F. R., and Powell, R. D.: Metallosomes. J. Struct. Biol., 127, 152-160 (1999).

    • Abstract (courtesy of Science Direct):
      LInk

Nanogold can also impart functionality. Hamad-Schifferli and co-workers used amino-modified molecular beacons labeled with Mono-Sulfo-NHS Nanogold to achieve remote control of DNA hybridization, via the highly localized temperature rise produced by the Nanogold® particle inductively coupled to a pulsed radio frequency magnetic field. Dubertret and colleagues have found Nanogold to be much more effective as a quencher in molecular beacons than the conventional DABCYL, improving the "signal-to-noise ratio" (the ratio of fluorescence intensity when the beacon is open to when it is closed) from 100 to up to several thousand.

References:

  • Hamad-Schifferli, K.; Schwartz, J. J.; Santos, A. T.; Zhang, S., and Jacobson, J. M.: Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna. Nature, 415, 152-155 (2002).

    • Abstract (Medline):
      LINK

  • Dubertret, B., Calame, M., and Libchaber, A.: Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol., 19, 365-370 (2001).

    • Abstract (Medline):
      LINK

More information:

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Nanogold® Labeling for Correlative Fluorescence and SEM

Many publications describe the use of Nanogold® for transmission electron microscopy, but it is also an effective label for scanning electron microscopy (SEM). The superior ability of Nanogold and FluoroNanogold for accessing targets was demonstrated recently by Elizabeth Schroeder-Reiter and colleagues, who describe the high-resolution detection and localization of phosphorylated histone H3 at serine 10 in mitotic barley chromosomes by correlative fluorescence and SEM. While a 10 nm colloidal gold conjugate gave poor labeling and lack of correlation with fluorescent signals, both the Nanogold and FluoroNanogold produced dense labeling which correlated well with both fluorescence labeling and known target distribution.

Barley chromosomes were isolated and mounted either on laser-marked glass slides or on standard glass slides. Chromosome control slides were fixed in 2.5% glutaraldehyde in cacodylate buffer (75 mM, pH 7); slides for immunolabeling were incubated in PBS (0.13 M NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4, pH 7.0 with 0.1% Tween 20). Labeling was done at room temperature; all wash steps were 3 x 10 min each. Slides were blocked with 1% bovine serum albumin in PBS for 30 min, then incubated with primary antibody, (polyclonal rabbit antibody against histone H3 phosphorylated at serine position 10) diluted 2 : 500 in the blocking solution for one hour. After washing in PBS, the secondary antibody (Cy3-labeled anti-rabbit diluted at 2 : 500 in blocking solution, anti-rabbit 10nm gold, anti-rabbit-FluoroNanogold or anti-rabbit-Nanogold diluted at 1 : 20 in blocking buffer) was applied for one hour.

The slides were subsequently washed in PBS, and specimens were routinely postfixed with 2% glutaraldehyde in PBS (without Tween 20). Immunogold-labeled specimens were washed with distilled water and either gold enhanced with GoldEnhance, or silver enhanced with HQ Silver. The slides were washed in 100% acetone, critical point dried (It was essential for preservation of three-dimensional chromosome ultrastructure and for viewing access of the metaphases with SEM that the slides were at no point allowed to air dry). Slides were first controlled with LM in phase contrast mode. They were carbon-coated by evaporation to a layer of 3 - 5 nm and examined at an accelerating voltage of 12 -15 kV with a Hitachi S-4100 field emission scanning electron microscope. Back-scattered electrons (BSE) were detected with a YAG-type detector (Autrata); secondary electron (SE) and BSE images were recorded simultaneously.

Reference:

Schroeder-Reiter, E.; Houben, A., and Wanner, G.: Immunogold labeling of chromosomes for scanning electron microscopy: A closer look at phosphorylated histone H3 in mitotic metaphase chromosomes of Hordeum vulgare. Chromosome Research, 11, 585-596 (2003).

Abstract (courtesy of Kluwer Academic Publishers):
LINK

More information:

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Gold Nanoparticle Biochips and Sensors

A while ago, we reported the use of Nanogold and silver enhancement as a simple, low-cost colorimetric detection method for biochips: Alexandre and co-workers compared the sensitivities of the Nanogold-silver colorimetric method with the Cy-3 fluorescence method, and found that the detection limits of both methods were equivalent and corresponds to 1 amol of biotinylated DNA attached on an array.

Reference:

Alexandre, I.; Hamels, S.; Dufour, S.; Collet, J.; Zammatteo, N.; De Longueville, F.; Gala, J. L., and Remacle, J. Colorimetric silver detection of DNA microarrays. Anal. Biochem., 295, 1-8 (2001).

Abstract (Medline):
LINK

An alternative, described recently by Fritzsche and group, is to use conductimetric detection of colloidal gold-labeled DNA. A gold-labeled target DNA, present in a test solution, was detected using a complementary capture DNA immobilized in a 1 micron microelectrode gap. A subsequent silver enhancement step was used to deposit conductive material onto the bound nanoparticles, and finally to produce an electrical contact between the electrodes. This detection scheme offers the potential for a simple low-cost, robust and highly miniaturizable method for biochip detection for point-of-care applications in the context of lab-on-a-chip technologies.

Reference:

Urban, M.; Mller, R., and Fritzsche, W.: A paralleled readout system for an electrical DNA-hybridization assay based on a microstructured electrode array. Rev. Sci. Instr., 74, 1077-1081 (2003).

Abstract (courtesy of the Review of Scientific Instruments):
LINK

Brainina and co-workers, meanwhile, report the preparation of an electrochemical immunosensor for the diagnosis of Forest-Spring encephalitis has been proposed, comprising a screen-printed thick-film graphite electrode serving as the transducer and a layer of the Forest-Spring encephalitis antigen immobilized on the electrode functioning as the biorecognition substance. Detection proceeds from the formation of an antigen-antibody immune complex, binding of colloidal gold-labeled protein A to the complex, and recording of the gold oxidation voltammogram, which provides information about the presence and concentration of antibodies in blood serum. The response is proportional to the concentration of antibodies over the interval from 107 to 102 mg/mL. The detection limit is 107 mg/mL.

Reference:

Brainina, K.; Kozitsina, A., and Beikin, J.: Electrochemical immunosensor for Forest-Spring encephalitis based on protein A labeled with colloidal gold. Anal. Bioanal. Chem., 376, 481-485 (2003).

  • Abstract (courtesy of SpringerLink):
    LINK

  • Reprint (PDF - courtesy of SpringerLink):
    LINK

More information:

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Other Recent Publications

Another choice of gold-conjugated nanobiotechnology building block was contributed recently by Wang and co-workers, in the form of cysteine-added mutants of cowpea mosaic virus; this can be produced readily in large quantities. The authors used Nanogold® to localize the thiols of cysteine residues engineered into the outer surface, then used formation of disulfide bonds, or derivatization with biocytin followed by streptavidin, to aggregate the virus particles in a controlled manner. The thiolated virus particles also bound to large (0.9 micron) gold particles.

Reference:

Wang, Q.; Lin, T.; Johnson, J. E.; and Finn, M. G.: Natural supramolecular building blocks. Cysteine-added mutants of cowpea mosaic virus. Chem. Biol., 9, 813-819 (2002).

Abstract (courtesy of Chemistry & Biology):
http://www.chembiol.com/content/article/abstract?uid=PIIS1074552102001667

Ziese and co-workers have demonstrated a new advantage of the high penetration of small gold conjugates into specimens. Using HAADF-STEM (high angular annular dark-field-scanning transmission electron microscopy) tomography, they were able to localize gold particles in three dimensions within sections of Epon-embedded, osmium-uranium-lead-stained biological material. Calculations showed that a 3D reconstruction obtained from HAADF-STEM projection images can be spatially aligned to one obtained from transmission electron microscopy (TEM) projections with subpixel accuracy.

Reference:

Ziese, U.; Kubel, C.; Verkleij, A., and Koster, A. J.: Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography. J Struct. Biol., 138, 58-62 (2002).

Abstract (Medline):
LINK

Saito and co-workers report promising results with new antigen retrieval procedures which confer improved stainability for postembedding immunogold electron microscopy. Weakly fixed cultured Helicobacter pylori (ATCC43504) were embedded in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on nickel grids and heated at 121°C for 15 min, 99°C for 40 min, and 65°C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65°C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. This suggests that the antigen retrieval method should be applied for routine use even by in postembedding immunogold electron microscopy.

Reference:

Saito, N.; Konishi, K.; Takeda, H.; Kato, M.; Sugiyama, T., and Asaka, M.: Antigen Retrieval Trial for Post-embedding Immunoelectron Microscopy by Heating with Several Unmasking Solutions. J Histochem. Cytochem., 51, 989-994 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/8/989

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