N A N O P R O B E S     E - N E W S

Vol. 9, No. 2          February 29, 2008

Jesús Tamarit-López and co-workers reported a novel technology for immunoassays in their recent paper in Analytica Chimica Acta: polystyrene spin-coated compact discs for microimmunoassaying, using silver-enhanced Nanogold as the detection system for the determination of a neurotoxic organophosphorous compound. Compact disks are highly suited to immunoassays: they are prepared using high-quality polymers, and are read using a laser which can be readily adapted to "read" chromogenic or metallographic signals. The assay format is a competitive immunoassay which in which the capture antibody is printed onto a CD; after incubation with the analyte, the CD is then treated with primary antibody followed by Nanogold-labeled secondary antibody and silver enhancement. It is then read using a 780 nm laser and detection board. The design of the assay is shown below:

The gold surface of the L-CD was conditioned by gently washing with 96° ethanol, then rinsing with deionized water. The disc was dried with slight centrifugation, then spun at 840 rpm, using a laboratory centrifuge. Next, 1 mL of 3% (w/v) polystyrene (PS) in Dowanol was dispensed with a pipette near the internal radius of the gold surface, so the polymer solution was evenly distributed over the entire surface while rotating for 1 minute at a constant speed. The coated CD was then heated in an oven at 60 C for 30 minutes in order to remove any remaining solvent.

The PS coated CD was coated with 35 µL of conjugate solution (OVA-triclopyr at 4 mg/L in coating buffer (50mM sodium carbonate buffer, pH 9.6) and evenly distributed using 22 mm 22 mm glass cover slips (eight sample areas per disc). The discs were then closed onto a CD box with a water-saturated filter paper for 16 hours at 4°C. The CD was then washed with deionized water for 1 minute and dried by slight centrifugation. Conjugate-coated discs prepared in this manner were stable for at least 3 months when stored at -20°C. Next, the primary antibody solution (C2-II: diluted 1 : 1600 in printing buffer, PBS-T: 10mM sodium phosphate buffer, 150mM NaCl, 0.01% (v/v) Tween 20, 5% (v/v) glycerol, pH 7.2), with or without chlorpyriphos, was arrayed onto the CD by stamping from a 384-well plate. After 5 minutes, the disc was washed with PBS-T for 1 minute, then rinsed with deionized water and dried as before. 35 µL of Nangold-IgG Goat anti-Rabbit IgG, diluted 1 : 100 in PBS-T, was dispensed onto each working area and covered with a glass cover slip. After an hour at room temperature, the disc was washed, rinsed and dried as before. For optical detection of the immunocomplex formation, the arrays were incubated with 50 µL of a 1:1 (v/v) mixture of silver enhancer solution for 12 minutes, covered as before. After washing with deionised water and drying, the disc was read.

The CD was read using a CD drive from Plextor America (Fremont, CA, USA), which has an optical system with a laser (lambda = 780 nm) to read standard CDs and uses the servo focus/tracking system to centre and focus the beam on the spiral data track of the entire disc surface. A planar photodiode SLSD-71N6 (Silonex, Montreal, Canada), 25.4 mm long and 5.04 mm wide, with a spectral range between 400 and 1100 nm, maximum spectral sensitivity of 0.55A/W at 940 nm, was attached to the upper part of the CD reader, 2mm above the L-CD, along the line scanned by the laser beam during the reading process. By varying the position of the photodiode in combination with the radial movement of the laser and rotation of the disc, almost the entire disk surface could be read. The electrical signal generated by the photodiode was digitalized by a 16-bit data acquisition board, saved in memory, then deconvoluted to give an image. The captured data are transferred to the computer through a USB 2.0 interface for quantification. The assay results were read using custom software written in Visual C+; this software also controlled the data acquisition board, selected the sampling frequency, and saved the resulting data to the computer hard disc in an uncompressed binary format.

The disc surface was large enough to simultaneously detect 8 separate assays per disc, or 2560 spots per disc. The detection limit for this organophosphorus compound was found to be 0.08 µgL^-1. These results show that this approach has enormous potential, and that polystyrene spin-coated compact discs used in combination with CD players can form the basis of an easy-to-operate, robust and portable devices for "lab-on-a-disc" analytical applications. No word, however, on what silver-enhanced Nanogold sounds like...

References:

Spin-coated compact disc immunoassay:

• Tamarit-López, J.; Morais, S.; Puchades, R., and Maquieira, Á: Anal. Chim. Acta, 609, 120-130 (2008).
  • Abstract (courtesy of Science Direct).

Nanogold with silver enhancement for biochips:

• 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 (courtesy of Science Direct).

More information:

• Nanogold conjugates: catalog and general information
• Nanogold conjugates: Product information and instructions
• Nanogold conjugates: technical help
• Nanogold labeling references, sorted by application

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What to Expect when Labeling Smaller Molecules with Nanogold®

Small molecules, particularly peptides and small proteins, can be particularly difficult to label successfully because they can demonstrate unexpected secondary interactions with the Nanogold reagents, and also because the solubility properties of the conjugates can be different to either of the reagents, and to what might be expected. Some of the issues that we have encountered with these molecules, and their solutions, are discussed below.

More information:

• Nanogold labeling reagents: catalog information
• Nanogold labeling reagents: technical help
• References: Nanogold labeling of peptides
• Guide to Gold Cluster labeling
• Nanogold labeling reagents: recent updates for better performance
• Strategies for separating and purifying Nanogold conjugates

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Nano-W Helps Find a Stable Cell Line for Generating Ebola Virus-like Particles

NanoVan and Nano-W are ideal for this because they have a highly amorphous structure and fine grain which provides for maximum resolution in specimen imaging, since crystallization can obscure features of interest. In addition, NanoVan is ideal for use with smaller gold labels such as Nanogold®: because the stain is less electron-dense than other negative stains such as uranyl acetate or lead citrate, contrast between the gold particles and their environment is preserved. It is very fine-grained and highly amorphous, and has been used for a number of high-resolution STEM and TEM studies of virus and protein ultrastructure. Nano-W gives a more dense stain, and is more suited to use with larger gold labels. NanoVan and Nano-W are based on organic salts of vanadium and tungsten respectively.

Advantages of these reagents:

• NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give any desired intermediate stain density.
• Near-neutral pH results in better ultrastructural preservation.
• NanoVan is less susceptible to electron beam damage than uranyl acetate.
• Fine grain allows high imaging resolution.

In a recent issue of the Journal of Virological Methods, Melito, Beniac and co-workers describe how Nano-W was used for the electron microscopic characterization of the production of Ebola virus-like particles by new cell lines expressing Ebola virus glycoproteins and the matrix protein VP40 utilizing an ecdysone inducible mammalian expression system. Ebolavirus is a filovirus that causes hemorrhagic fever in humans, with fatality rates of up to 90%. The lack of therapeutic interventions, combined with the threat of weaponizing this organism, makes understanding the organism and developing prevention and therapeutic responses a priority.

The expression of key viral proteins and the production of virus-like particles in mammalian systems are often pursued as a part of characterization and functional studies. Commonly, these proteins are expressed through transient transfection of mammalian cells. Unfortunately, the transfection reagents are expensive and the process is time consuming and labor-intensive. The creation of stable mammalian cell lines expressing EBOV proteins and producing EBOV VLPs would address the limitations of current technology and provide an expression system with the properties necessary to study and use these proteins. The authors used an ecdysone inducible mammalian expression system to create stable cell lines that express the Ebolavirus transmembrane glycoprotein (GP), the soluble glycoprotein (sGP) and the matrix protein (VP40) individually as well as GP and VP40 simultaneously (for the production of virus like particles).

Expression products were analyzed by Western blotting and electron microscopy. Cell culture supernatant was harvested from a T-150 tissue culture flask. VLP and glycoprotein virosome were purified by centrifugation at 500g for 10 minutes at 4°C to remove cell debris. The clarified supernatant was then layered over an 8 mL 20% sucrose cushion in 25 mm 89 mm Ultra Clear centrifuge tubes and centrifuged using a SW28 rotor for 2 hours at 28,000 rpm. After the supernatant and sucrose cushion were then poured off, the pellet resuspended in 20mM Tris, 0.1M NaCl, 0.1mM EDTA, pH 7.4 and centrifuged under identical conditions for 30 minutes. The final pellet was resuspended in 0.5 mL of 20mM Tris, 0.1M NaCl, 0.1mM EDTA, pH 7.4. sGP was purified by fast protein liquid chromatography (FPLC) with desalting and anion exchange columns. All peaks were analyzed by Western blot using mouse anti-EBOV sGP antibody. The sGP eluted at approximately 30% elution buffer. Fractions were collected and concentrated using an Amicon Centricon-4 centrifuge filter, molecular weight cut off of 30 kDa, prior to analysis.

For electron microscopy, the EBOV VLPs and glycoprotein virosome samples were adsorbed to a carbon coated formvar film on a 400 mesh copper grid for 1 minute, washed in PBS (3 x 1 minute), followed by fixation for 2 minutes (1% paraformaldehyde, 2% glutaraldehyde in PBS). Grids were then washed in deionised water and negatively contrasted with Nano-W. Specimens were imaged in a transmission electron microscope (TEM) operating at 200 kV, and at nominal instrument magnifications of 14,500 x and 50,000 x. Digital images of the specimens were acquired by CCD camera. Western blot analysis and electron microscopy. They were confirmed to be the same as those expressed by the transient system.

The inducible system was shown to be a significant improvement over current technology: it simplifies the process, and provides a method for more cost-effective production of viral proteins and even virus-like particles for study. This promises to be a valuable tool for the study of viral pathology and for the development of methods for preventing and treating viral infections.

Reference:

• Melito, P. L.; Qiu, X.; Fernando, L. M.; Devarennes, S. L.; Beniac, D. R.; Booth, T. F., and Jones, S. M.: The creation of stable cell lines expressing Ebola virus glycoproteins and the matrix protein VP40 and generating Ebola virus-like particles utilizing an ecdysone inducible mammalian expression system. J. Virol. Methods, 148, 237-243 (2008).
  • Abstract (courtesy of Science Direct).

More information:

• Catalog information: Negative stain reagents
• NanoVan: Product information and instructions
• Nano-W: Product information and instructions
• Technical help: Negative staining
• NanoVan Negative stain: Our paper from Microscopy and Microanalysis 1994
• Negative staining: References

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Nanogold® with HQ Silver: Ideal for Pre-Embedding EM for Neuroscience

• High penetration into cells and tissues.
• High labeling density.
• Close to quantitative labeling of antigenic sites.
• High labeling resolution.

Together with HQ Silver, this provides the ideal combination of features to produce high-quality staining:

• The only commercial silver enhancer to feature a protective colloid for highest size uniformity and morphologically consistent enhancement.
• Near neutral pH and low ionic strength ensure maximum specimen integrity.
• High proportion of gold particles are enlarged.
• Highly selective reaction with low background.

This month is no exception. Contributing to the extensive applications of this month, Furuta and group, in a recent issue of the Journal of Neuroscience, describe the use of Nanogold with HQ Silver enhancement as one method, together with electrophysiological recordings, retrograde labeling with in situ hybridization, and anterograde labeling with immunoelectron microscopy, to explore the morphology and nature of cellular connections in trigeminal sensory nuclei, the first processing stage in the vibrissal system of rodents, from the brains of Sprague-Dawley rats.

Trigeminal sensory nuclei feature separate populations of thalamic projecting cells and a rich network of intersubnuclear connections. The signals conveyed to the cortex by each of the ascending pathways of vibrissal information depends on local transactions that occur in the brainstem. The authors began by using electrophysiological measurements to determine whether intersubnuclear projections from the spinal complex could inhibit vibrissal responses in the principal trigeminal nucleus (PrV). To address this question, a single-whisker flutter stimulator was constructed; Once a Principal whisker (PW) was identified, it was isolated from the surrounding whiskers so that electophysiological measurements could be made of the response to stimulation of this whisker alone. A marked reduction in the magnitude of vibrissal responses was observed in the PrV after unilateral electrolytic lesion of the SpVi. In normal rats, population poststimulus time histograms (PSTH) in response to an air jet exhibited a transient peak of excitation at stimulus onset, followed by an abrupt decline during the stimulus plateau. In SpVi-lesioned rats, the early peak of excitation was still prominent, but the decline of sustained discharges was markedly attenuated; this result suggested that the SpVi lesion might have severed intersubnuclear connections that exert an inhibitory action in the PrV.

The authors proceeded to investigate the distribution of glutamatergic and GABAergic neurons in the trigeminal nuclei using in situ hybridization for glutamate- and GABA-related specific transcripts. In situ hybridization for VGluT1 labeled large-sized neurons throughout the SpVi, a dense population of small-sized cells in the PrV, and a smaller population of large cells in the rostromedial part of the PrV and in the neighboring infratrigeminal nucleus; the oralis subnucleus (SpVo) and caudalis subnuclei (SpVc) contained the lowest density of cells expressing this transcript. A dense population of small-sized PrV cells expressed VGluT2, which was also strongly expressed by large-sized neurons in the SpVo and in the rostral sector of the SpVi. The density of VGluT2-positive cells decreased in the caudal part of the SpVi, and was moderate in the SpVc. In situ hybridization for the GAD and VIAAT mRNAs labeled a high density of small cells in the caudal sector of the SpVi and in the SpVc, and a moderate number of cells in the SpVo. The lowest expression of both GABA-related transcripts was observed in the PrV. These results show that the different subnuclei that give rise to the ascending vibrissal pathways contain different subtypes of glutamatergic cells and different density of GABAergic neurons. anterograde labeling of intersubnuclear projections was achieved by pressure injection in the SpVi of 0.5 µl of GFP-expressing recombinant Sindbis virus (2 x 10⁹ infectious U/ml). Retrograde tracing experiments showed that intersubnuclear projections to the PrV arise from different populations of GABAergic and glutamatergic neurons that are principally located in the caudal sector of the SpVi and in the SpVc respectively. In accord with the results of in situ hybridization in rats, the PrV in GAD-GFP mice contained very few GFP-positive cells, whereas the SpVi contained the most; Fluorogold injection into the PrV of three GAD-GFP mice also revealed a number of doubly labeled cells in the SpVi. This confirms that, as in rats, the SpVi in mice is at the origin of a significant GABAergic projection to the PrV.

Electron microscopic labeling was then used to characterize the ultrastructure of the synaptic contacts established by interpolaris axons in the PrV was studied in six rats, by examining 101 vesicle-containing terminals that had been anterogradely labeled with a GFP-expressing recombinant Sindbis virus injected into the caudal sector of the SpVi. Brains that received virus injection were cut into 50-µm-thick horizontal sections on a vibratome. The resulting sections were incubated in phosphate-buffered saline (PBS) containing 20% normal donkey serum and 0.2% Photo-Flo, then incubated overnight with anti-GFP guinea pig antibody (0.05 µg/µL) in PBS containing 2% normal donkey serum and 0.2% Photo-Flo at 4°C. After washing with PBS, sections were then incubated overnight with 1:1000 diluted gold-conjugated anti-guinea pig IgG goat antibody in PBS containing 2% normal donkey serum at 4°C, post-fixed in 2% glutaraldehyde in PBS, then silver intensified with HQ Silver. All sections were treated with OsO₄ (1% for 1 minute, then 0.5% for 20 minutes at 4°C) in phosphate buffer, dehydrated in ethanol and propylene oxide, and embedded in Durcupan. During dehydration, sections were treated with 1% uranyl acetate in 70% ethanol for 40 minutes. Ultrathin sections were cut with an ultramicrotome and mounted on nickel grids. Postembedding GABA immunostaining was performed using enzymatic immunohistochemistry. Ultrathin sections were observed with an electron microscope (H-7100; Hitachi, Tokyo, Japan). Seventy-six of the terminals exhibited GABA immunoreactivity, of which 43 made symmetric (n = 40) or asymmetric (n = 3) synaptic contacts with dendrites. Two of the 76 terminals established axoaxonic contacts with GFP-negative large terminals that contained many mitochondria and made asymmetric synapses with dendrites; these are consistent with inhibitory contacts.

Together, the results from these different approaches provide conclusive evidence that the principal trigeminal nucleus receives inhibitory GABAergic projections from the caudal sector of the interpolaris subnucleus, and excitatory glutamatergic projections from the caudalis subnucleus. These results suggest the possibility that, by controlling the activity of intersubnuclear projecting cells, brain regions that project to the spinal trigeminal nuclei may take an active part in selecting the type of vibrissal information that is conveyed through the lemniscal pathway.

Reference:

• Furuta, T.; Timofeeva, E.; Nakamura, K.; Okamoto-Furuta, K.; Togo, M.; Kaneko, T., and Deschênes, M.: Inhibitory gating of vibrissal inputs in the brainstem. J. Neurosci., 28, 1789-1797 (2008).
  • Abstract (courtesy of the Journal of Neuroscience).

More information:

• A covalent 10 nm gold immunoprobe: Our paper from Microscopy and Microanalysis 1999
• Towards Bigger Nanogold:: Our paper from Microsopy and Microanalysis 2005
• 5 nm Gold-Ni-NTA binds His Tags: Our paper from Microscopy and Microanalysis 2005
• Guide to Gold Cluster Labeling: separation of conjugates
• Other product and research applications

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New Catalog: Ask for Your Copy Now!

The last few years have seen an explosion of new applications for gold nanoparticles in nanotechnology applications, and Nanogold and related products have been featured in many exciting publications in these fields. Our new catalog includes the key references and shows how the key features of Nanogold, such as its specific reactivity, solubility, and site-specific conjugation chemistry, have helped enable these novel uses.

We will be sending out our new catalog with fulfilled orders: however, if you'd like a copy but you are not yet ready to place your order, please call us at 1-877-447-6266, or e-mail us to request yours.

Long Island Regional SBIR Workshop: Nanoprobes will be participating in a panel discussion on obtaining SBIR grants, a valuable funding mechanism for technology-based startups and small businesses, at the Long Island Regional Small Business Innovation Research (SBIR) Workshop, hosted by the Stony Brook Center for Biotechnology on March 20. Look for us in the panel discussion to be held towards the end of the afternoon, and feel free to ask questions afterwards.

More information:

• Contact information
• Nanoprobes: news and press releases
• About Us: company information
• Long Island Regional Small Business Innovation Research (SBIR) Workshop

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Other Recent Publications

Reference:

• Sokolova, V., and Epple, M.: Inorganic nanoparticles as carriers of nucleic acids into cells. Angew. Chem. Int. Ed. Engl.,, 47, 1382-1395 (2008).
  • Abstract (courtesy of the Wiley-Interscience).

Ankona Datta and co-workers, in a recent Journal of the American Chemical Society, report the preparation of high relaxivity, macromolecular contrast agents based on the conjugation of gadolinium chelates to the interior and exterior surfaces of MS2 viral capsids. The proton nuclear magnetic relaxation dispersion (NMRD) profiles of the conjugates show up to a 5-fold increase in relaxivity, leading to a peak relaxivity (per Gd³⁺ ion) of 41.6 mM^-1s^-1 at 30 MHz for the internally modified capsids. Modification of the exterior was achieved through conjugation of a Gd³⁺ chelating ligand suitably functionalized for selective bioconjugation, prepared by attaching an alkoxyamine linker to a heteropodal TREN-bis-HOPO-TAM ligand, to flexible lysines via aldehyde modification; internal modification was accomplished by conjugation to relatively rigid tyrosines. Higher relaxivities were obtained for the internally modified capsids, showing that there is facile diffusion of water to the interior of capsids, and also demonstrating that the rigidity of the linker attaching the complex to the macromolecule is important for obtaining high relaxivity enhancements. The viral capsid conjugated gadolinium hydroxypyridonate complexes appear to possess two inner-sphere water molecules. These results indicate that there are significant advantages of using the internal surface of the capsids for contrast agent attachment, leaving the exterior surface available for the installation of tissue targeting groups.

Reference:

• Datta, A.; Hooker, J. M.; Botta, M.; Francis, M. B.; Aime, S., and Raymond, K. N.: High relaxivity gadolinium hydroxypyridonate-viral capsid conjugates: nanosized MRI contrast agents. J. Amer. Chem. Soc., 130, 2546-2552 (2008).
  • Abstract (courtesy of the ACS Publications).

Xiong, Gang and group added their take on the construction of nanoparticle-DNA hybrids in another recent communication in the Journal of the American Chemical Society. DNA-conjugated gold nanoparticles were hybridized with linker DNAs, whose two ends are complementary to the respective mutually noncomplementary ssDNAs on the nanoparticles. This approach is attractive because it allows the building various architectures from a given set of functionalized nanoparticles using different linker designs. A binary set of gold nanoparticles of diameter 11.5 ± 1.1 nm was generated by functionalizing colloids with either type A or B of noncomplementary ssDNA: (A) 5'-ATTGGAAGTGGATAA-(T)15-C₃3H₃₆3-SH; or (B) HS-C₆H₁₂-(T)15-TAACCTAACCTTCAT-3'). The ssDNA contains a 15 base-pair (bp) outer recognition part and a 15 bp poly dT, which acts as a spacer separating the recognition sequence from the gold surface. The ends of the linker Ln ssDNAs (5'-TTATCCACTTCCAAT-(T) n-ATGAAGGTTAGGTTA-3') are complementary to the respective ends of ssDNAs on AuNCs and are separated by a central flexible poly-dT fragment which contains n nucleotides, having a length of 0, 15, 30, or 70 bp. Each system was formed by mixing an equal mole of the two types of ssDNAs capped gold nanoparticles and a linker ssDNA (DNA/particle mole ratio 36:1) in 0.3 M phosphate-buffered saline (PBS) buffer. The mixture was incubated at 65°C for 10 minutes, then cooled to room temperature for about 2 hours. Temperature-dependent synchrotron small-angle X-ray scattering (SAXS) is utilized to characterize the structure of assembled aggregates; different crystal packing was found in the resulting assemblies, depending on the temperature and linker length. Formation of crystalline organization was observed to be favored for longer ssDNA linkers.

Reference:

• Xiong, H.; van der Lelie, D., and Gang, O.: DNA linker-mediated crystallization of nanocolloids. J. Amer. Chem. Soc., 130, 2442-2443 (2008).
  • Abstract (courtesy of the ACS Publications).

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