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N A N O P R O B E S     E - N E W S

Vol. 10, No. 3          March 27, 2009


In this Issue:


This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful, as always, let us know if we can improve anything.

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EnzMetTM: Better for In Situ Hybridization, ideal for Microelectrode Biochips

Chromogenic in situ hybridization (CISH) is fast becoming an accepted alternative to fluorescent in situ hybridization (FISH). Nanoprobes has developed a number of detection technologies for CISH that provide higher sensitivity, lower copy number detection and clearer signals than enzyme chromogens such as DAB, including Nanogold® with silver acetate autometallography and Nanogold with gold enhancement (GOLDFISH). However, the best method yet is EnzMetTM, or enzyme metallography. EnzMet provide higher sensitivity, virtually zero background, and allows you to count individual gene copies - all major advantages over previous methods.

EnzMetTM for in situ hybridization (ISH)
and Microelectrode Biochips

EnzMetTM (enzyme metallography) is a detection and staining method in which a targeted enzymatic probe is used to selectively deposit metal from solution. For applications in optical microscopy and optical detection, it provides important advantages over conventional organic enzyme chromogen such as DAB. Advantages include:

  • EnzMetTM technology uses HRP to deposit metallic silver with extraordinary selectivity. Background is virtually zero.
  • High sensitivity: detect single copies of target genes, or low-abundance proteins with almost no background.
  • Virtually no diffusion of reaction product means super-sharp signals with highest resolution. Count individual gene copies.
  • Black, sharply defined, non-diffusing stain lets you clearly see underlying morphology. EnzMetTM is readily distinguished from all counterstains.
  • Does not fade or bleach: can be archived indefinitely.

For clinical applications, EnzMetTM has been licensed to Ventana Medical Systems (now part of Roche) for use as a detection system in automated slide stainers; it is marketed and sold as SISH (Silver In Situ Hybridization). It is already available in Europe and other parts of the world, and is currently awaiting FDA approval in the USA. However, if you are doing in situ hybridization without an automated slide staining platform, you can purchase EnzMetTM reagents from us for research use.

For in situ hybridization, use EnzMetTM HRP Detection Kit for IHC/ISH (catalog number 6001) from Nanoprobes. Complete product information, instructions and protocols for this reagent are available on our web site.

Reference:

  • Powell, R. D.; Pettay, J. D.; Powell, W. C.; Roche, P. C.; Grogan, T. M.; Hainfeld, J. F., and Tubbs, R. R.: Metallographic in situ hybridization. Hum. Pathol., 38, 1145-1159 (2007).

Different Nanogold and metallographic in situ hybridization methods are compared with the conventional organic chromogen, diaminobenzidine (DAB) below: you can see the evolution of metallographic chromogenic in situ hybridization, and the improvements provided by EnzMetTM.
[Metallographic in situ hybridization results (129k)]
(a) and (b): Single copies of HPV-16 in SiHa cells detected by tyramide signal amplification (TSA, also known as CARD, or catalyzed reporter deposition) followed by detection with (a) streptavidin-peroxidase developed with DAB, and (b) streptavidin-Nanogold with silver acetate autometallography. Copies of HPV-16 appear as single spots. (H & E counterstain. Original magnification X 560). (c) Nanogold with gold enhancement (GOLDFISH) procedure in tissue with HER2 gene amplification, showing large, confluent nuclear signals from multiple gene copies in close proximity. Nuclear fast red counterstain (original magnification 400). (d): EnzMetTM detection of the amplification of individual HER2 gene copies in paraffin-embedded human invasive breast carcinoma biopsy; normal, non-amplified cells contain two copies of the HER2 gene, while the infiltrating HER2-amplified carcinoma cells show multiple copies (original magnification X 400. Image courtesy of Dr. R. R. Tubbs, Cleveland Clinic Foundation). (e) design of electrical detection element for use on biochips: target oligonucleotide is deposited between electrodes, then bound by a complementary enzymatic probe which is "developed" with enzyme metallography to produce a conductive connection.


EnzMetTM for immunohistochemistry (IHC) and blotting

However, EnzMetTM is not just for optical detection of in situ hybridization. It is also highly effective for electrical detection of DNA on biochips, where it provides improvements over silver-enhanced colloidal gold for the electrical detection of target DNA using arrays of electrodes: the principle is shown above. Now, in their recent paper in Biosensors and Bioelectronics, Schüler, Fritzsche and co-workers demonstrate that this process can be used on screen printed electrode structures for chip-based electrical detection of viral DNA (150 bp PCR products of human CMV DNA). These electrode arrays were produced on a glass substrate, making an additional optical readout possible. The screen printed structures showed the required precision, and are compatible with the applied biochemical protocols. When compared with chip substrates produced by standard photolithography, the screen printed chips showed the same sensitivity and specificity. Therefore, screen printing of electrode arrays for DNA chips for electrical detection provides an interesting and cost-efficient method for the production of DNA chips with microstructured electrodes.

If you wish to try this application for yourself, you should use EnzMetTM HRP Detection Kit for Research Applications (catalog number 6010) from Nanoprobes. Complete product information, instructions and protocols for this reagent are available on our web site.

Reference:

  • Schüler T, Asmus T, Fritzsche W, Möller R.: Screen printing as cost-efficient fabrication method for DNA-chips with electrical readout for detection of viral DNA. Biosens. Bioelectr., 24, 2077-2084 (2009).

EnzMetTM is also the ideal detection and visualization method for immunohistochemistry (IHC), and it can be used for highly sensitive detection with minimal background and very high contrast on blots. Immunohistochemical application was tested using high-complexity tissue microarrays (TMAs). 88 common solid tumors were evaluated by enzyme metallography (EnzMet) using an automated slide staining system (Ventana Medical Systems); targets were chosen to assess the ability of EnzMet to specifically localize encoded antigens in the nucleus (estrogen receptor), cytoplasm (cytokeratins), and cytoplasmic membrane (HER2) in TMAs. The intensity of staining for all three antigens evaluated was comparable for breast tumors as well as carcinomas of kidney, colon, and prostate. However, the quality of staining in EnzMet IHC preparations was much sharper, and the stain deposits were better defined, showing a more punctate appearance, than that found with DAB. Full concordance was found between the EnzMet and conventional IHC results. The EnzMet reaction product was dense and sharply defined, did not appreciably diffuse, and provided excellent high-resolution differentiation of cellular compartments in paraffin sections for the nuclear, cytoplasmic, and cell membrane-localized antigens evaluated. The higher density of elemental silver deposited during enzyme metallography permitted evaluation of core immunophenotypes at a relatively low magnification, without the need for oil immersion, allowing more tissue to be screened in an efficient manner. In addition, the signal is stable and provides a permanent record.

Reference:

  • Tubbs R.; Pettay J.; Powell R.; Hicks D. G.; Roche P.; Powell W.; Grogan T., and Hainfeld, J. F.: High-resolution immunophenotyping of subcellular compartments in tissue microarrays by enzyme metallography. Appl. Immunohistochem. Mol. Morphol., 13, 371-375 (2005).

For immunohistochemistry, use EnzMet EnzMet HRP Detection Kit for IHC/ISH (catalog number 6001) from Nanoprobes. Complete product information, instructions and protocols for this reagent are available online.

[EnzMet for IHC and Western blot (60k)]

Left: Immunoperoxidase staining of epithelial cytokeratins in paraffin-embedded human bladder tumor:
(a) secondary immunoperoxidase with DAB; and (b) secondary immunoperoxidase method using EnzMetTM (original magnification x 400).

Right: Comparison of Western blot detection of his-tagged Fusion Protein using HRP conjugates developed with (c) DAB and (d) EnzMetTM.

After transfer, membranes were incubated with anti-His-Tag (6xHis) monoclonal antibody, followed by BSA blocking, and then exposed to Horseradish peroxidase (HRP)-conjugated secondary antibody. The His-tagged fusion proteins were then visualized by DAB detection (Panel A) or EnzMetTM detection (Panel B). Lanes 1 and 4: 0.1 µg of 34 kDa his-tagged ATF-1. Lanes 2 and 5: 0.1 µg of 68 kDa his-tagged YY1. Lanes 3 and 6: 0.1 µg BSA and 0.1 µg ovalbumin.

In most cases, the EnzMetTM protocol using EnzMetTM for Blots below may be substituted for conventional DAB development without further modification of the protocol. However, because of its greater sensitivity, greater dilutions of either primary antibody or secondary probes may be required to achieve the optimum combination of sensitivity and clarity. A five-fold to ten-fold additional dilution has been found to give good results in immunohistochemical experiments and is likely to be appropriate here also.

Protocol:

  1. Wash with buffer containing 0.1% Tween-20 for 3 x 5 minutes.
    Note: Phosphate buffered saline, tris buffered saline or other wash buffers can be used. Including 0.1 % (w/v) Tween-20 in the wash buffer was found to be helpful in reducing non-specific binding.

  2. Wash with deionized water for 3 x 5 minutes.

  3. Shake off excess water. Cover membrane with 6 mL (or 3 volumes) of EnzMetTM Detect A. Incubate for 4 minutes.
    Note: Excess water can lead to the dilution of EnzMetTM reagents, resulting in weak staining and results which are difficult of reproducing.

  4. Add 2 mL (or 1 volume) of EnzMetTM Detect B to the membrane, and gently mix Solutions A and B. Incubate for 4 minutes.

  5. Add 2 mL (or 1 volume) of EnzMetTM Detect C to the membrane, and gently mix Solutions A, B and C. Incubate for 9 - 25 minutes, or until satisfactory staining is achieved.
    Note: The EnzMetTM incubation time mainly depends on the target concentrations and staining temperature. Longer incubation may be needed for visualizing low concentration targets. However, longer incubation may lead to some non specific background staining. The variation of EnzMetTM staining temperature can affect its silver deposition rate. Lower temperature slows down the deposition process, and thus a longer staining time may be required to reach a certain degree of staining density and sensitivity.

  6. Wash with deionized water for 3 x 5 minutes.

  7. Air dry membrane for record.

For Western and other blotting applications, you should use EnzMetTM EnzMet Western Blot HRP Detection Kit (catalog number 6002) from Nanoprobes. Complete product information, instructions and protocols for this reagent are available online.

Reference:

  • Liu, W.; Mitra, D.; Powell, R.; Tubbs, R.; Pettay, J., and Hainfeld, J.: Enzyme Metallography Silver Deposition for HRP Detection. Presented at ASCB 2007, Washington DC, December 1-4, 2007,: Poster # B349, Presentation # 1209.

More information:

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Dispensing HQ Silver,
and Controlling Nanogold® Stoichiometry

Two more topics from our technical support files:

Solving a sticky situation:
Handling HQ Silver

HQ Silver is a unique silver enhancement reagent. It contains a thickening agent which is a natural polymeric gum: this is used to modulate the rate of reaction of the mixture with gold nanoparticles. This gives the reagent a number of advantages, which result in improved performance compared with other silver enhancers, as illustrated below:

  • The rate of development of particles is very uniform, and once enlarged, the silver-enhanced gold particles have a high degree of size monodispersity.

  • The pH of the mixed reagent is close to neutral. This means that pH effects on tissues and cells are minimalized, and also that the reagent reacts with the highest possible proportion of gold particles. Although it develops more rapidly than LI Silver or some other silver enhancement reagents (typical development times are from 1 to 8 minutes), the reaction is controlled and precise, and final particle size may be adjusted by changing development time.

  • Ionic strength is very low. This ensures minimal effect on specimen ultrastructure, and hence excellent tissue preservation.

[Pre-embedding labeling with Nanogold-Fab' and HQ Silver (133k)]
Pre-embedding immunoelectron microscopy with Nanogold® and HQ Silver. Nanogold-Fab' goat anti-rabbit IgG secondary antibody labeling the K+ channel Kv2.1 subunit in rat brain, followed by HQ Silver enhancement. Note the high density and specificity of immunostaining, even elucidating subunit localization to cytoplasmic side of cell membrane and outer stacks of the Golgi; axons and terminals are clearly negative. Work done by J. Du, J.-H. Tao-Cheng, P. Zerfas, and C. J. McBain, NIH. See Neuroscience, 84, 37-48 (1998). Bar = 1 micron.

These features make HQ Silver ideal for electron microscopy, but because of the presence of the natural thickening agent, the components are more vulnerable to microbial contamination than some other silver enhancement reagents, and their viscous nature means that you may need to use a little more care in working with this reagent. When using HQ Silver, you will obtain the best results using these tips and tricks:

  • HQ Silver must be stored under conditions that do not allow microbial contamination and growth. If you plan not to use the reagent for more than a few days, the components should be stored frozen, preferably at -20°C.

  • Repeated freeze-thaw cycles can degrade the performance of the thickening agent. Therefore, when you first use HQ Silver, you will obtain the best results over the lifetime of the reagent if you divide each component into aliquots, each sufficient for your typical experimental run, then freeze the unused aliquots separately. Then, each time you conduct an experiment or a series of experiments that requires HQ Silver, thaw and mix one set of aliquots.

  • Because the components include different amounts of the thickening agent, they have different viscosities, and if they are dispensed quickly, it can be challenging to dispense equal amounts. You will obtain best results using an adjustable pipette set to draw and dispense the required amount, using a different tip for each component; drawing slowly will help avoid air bubbles. A positive-displacement pipette is best. A slow manual draw is actually preferable to an automatic draw since it will result in a more accurate dispense with viscous solutions. Component B ("Moderator") actually does not contain either of the components necessary for silver deposition: therefore, you will ensure the shortest delay between mixing and adding to your specimen if you dispense B first, then A and C.

  • Solvents and buffers used for our products are degassed before use to eliminate dissolved oxygen and the risk of formation of bubbles. Should it be necessary to agitate the solutions, use a vortexer on a low setting rather than agitating by hand; if agitating gently for an extended period (such as with a blot or slide), use a rotating shaker on a low setting - these will introduce fewer bubbles than manual agitation.

For light microscopy and blotting applications where ultrastructural preservation and uniform particle size are not an issue, you should consider using LI Silver instead. This reagent develops more slowly, and since it is colorless and non-viscous, development can be monitored more easily by light microscopy or other optical detection methods. Alternatively, gold enhancement may give better results in some applications.

More information:

Controlling stoichiometry: Nanogold® to biomolecule ratios

We are asked quite frequently how you control the stoichiometry of Nanogold® labeling. Although our Nanogold labeling reagents are formulated to contain close to one reactive functionality per gold particle, if the biomolecule you wish to label has more than one reactive group, you may still need to consider how to avoid attaching multiple golds - particularly if you also wish to easily isolate your conjugate product. The following points will help you achieve the most precise labeling:

  • Our Monomaleimido Nanogold, Mono-Sulfo-NHS-Nanogold and Monoamino Nanogold reagents are all formulated to contain close to one reactive functionality per gold particle; this is achieved through the separation of Monoamino Nanogold (the starting material for the other two reagents) over an anionic ion exchange column, which separates gold particles with different numbers of amino- groups, and in our labeling experiments, these reagents behave in a monofunctional manner. Each Nanogold particle will therefore react with only one biomolecule.

  • When labeling with Nanogold, you should usually select a reaction that labels a unique functional group- i.e. one that occurs only once in your biomolecule, such as a cysteine, rather than a functional group that is present at several different sites.

  • Size matters for separation: Nanogold has a molecular weight close to 15,000, but it may behave similarly to a smaller protein because the majority of its mass is from heavy gold atoms. When you label a larger biomolecule such as an antibody or protein, we recommend using a small excess of Nanogold because chromatographically, it is usually easier to separate excess smaller gold from the conjugate product than it is to separate unlabeled protein. However, since you would normally only use between 1.5 and 3 Nanogolds per biomolecule and the reaction yield is usually less than 100%, this imposes a relatively low upper limit on the number of Nanogold labels per protein.

  • When labeling molecules that are similar in size to Nanogold, or smaller, you would use a ratio of Nanogold : biomolecule of close to 1 : 1, or for smaller molecules, an excess of the smaller molecule. Since the Nanogold is monofunctional, this restricts most labeling to one Nanogold per biomolecule even if the biomolecule has multiple binding sites.

  • The Nanogold label is quite large: including its coordinated ligands, it has an overall molecule diameter of about 2.6 nm. Once attached, it hinders the approach of additional Nanogold reagents to nearby sites. This also favors close to a 1 : 1 ratio of Nanogold : conjugate biomolecule.

  • Nanogold is not charged, but it is soluble - it is functionalized in a manner that provides solubility, so it is dissolved rather than being merely suspended. Therefore, although there is no formal repulsion between Nanogold particles in solution (unlike colloidal gold) the affinity of the Nanogold particle surface for aqueous solution ensures that they remain dissolved and separated.

One exception to this rule is NTA-Ni(II)-Nanogold, which has multiple nitrilotriacetic acid-nickel (II) functionalities in order to increase binding to polyhistidine tags through the chelate effect obtained by multiple NTA-Ni(II) : His binding. In this case, control of stoichiometry is usually achieved by using an excess of Nanogold over the His-tagged biomolecule: for a biomolecule bearing a unique His tag, the excess of Nanogold favors the formation of 1 : 1 conjugates, both because the His-tagged biomolecule has a higher probability of encountering unconjugated Nanogold, and because conjugated Nanogold is sterically hindered by the bound molecule. Should you wish to use Positively Charged Nanogold or Negatively Charged Nanogold for labeling or conjugation, similar considerations apply.

More information:

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NTA-Ni(II)-Nanogold®:
New Technology for High-Resolution EM and Blots

Ni-NTA-Nanogold® is a new type of gold probe in which the targeting agent is not an antibody or protein, but the metal chelate nitrilotriacetic acid (NTA) nickel (II), which binds highly selectively to polyhistidine (His) tags. Because His tags may be readily engineered into expressed proteins, NTA-Ni(II)-Nanogold can be used to localize a wide variety of recombinant His-tagged proteins, and because it is much smaller than an antibody, it provides much higher resolution.

[Ni-NTA-Nanogold structure and STEM (48k)]
Left: Structure of Ni-NTA-Nanogold, showing the binding of the incorporated metal chelate to a His-tagged protein. Inset shows the resolution, expressed as the distance from the center of the Nanogold particle to the His tag: note that this is significantly shorter than the equivalent distance with antibodies. Right: Knob protein from adenovirus cloned with 6x-His tag, labeled with Ni-NTA-Nanogold, column purified from excess gold, and viewed in the scanning transmission electron microscope (STEM) unstained (Full width approximately 245 nm).

Ni-NTA-Nanogold™ has several significant advantages over conventional antibody probes:

  • Better penetration: because it is so small, NTA-Ni(II)-Nanogold can more easily penetrate into specimens and access sterically restricted sites within specimens, and perturbs their ultrastructure less. In some systems it may be used with stronger fixation or less permeabilization, enabling labeling with better ultrastructural preservation.
  • Higher labeling resolution. The nitrilotriacetic acid - Ni(II) chelate is much smaller than an antibody or protein, and therefore when it is bound, the gold is much closer to its target. This makes NTA-Ni(II)-Nanogold ideal for localizing sites in protein complexes or other macromolecular assemblies at molecular resolution.
  • NTA-Ni(II)-Nanogold is prepared using a modified gold particle, with very high solubility and stability. At 1.8 nm in size, it is readily visualized by electron microscopy.
  • Binding constants for Ni(II)-NTA are very high due to the combination of the chelate effect of multiple histidine binding, and target binding of multiple Ni(II)-NTA functionalization. Dissociation constants are estimated to be between 10-7 to 10-13 M-1. For many applications, this provides binding strengths comparable to antibodies.

Applications include:

  • High-resolution labeling of proteins, protein complexes or organelles containing recombinant His-tagged proteins for EM1 or STEM2 localization.
  • Labeling and molecular localization of two different subunits of Photosystem II.
  • "Universal" pre-embedding labeling of His-tagged proteins in tissue sections for electron microscopic observation.
  • identifying His-tagged proteins in fractions during Ni-NTA-column purifications.
  • Detection of recombinant His-tagged proteins on blots and in gels.
  • Heavy atom labeling of regular structures for image analysis and structure solution.

Ni-NTA-Nanogold® has found extensive application in cryoelectron microscopy. One example, included in a recent review on polysaccharide export systems in gram-negative bacteria, is the localization of domains within Wzc, a tyrosine autokinase with a central role in the coordinated biosynthesis and secretion process of the polysaccharides that make up the capsule that protects encapsulated Escherichia coli bacteria from host immune defenses. The capsule is formed from K antigenic capsular polysaccharide: its assembly and translocation require proteins in the inner and outer membranes, and the inner membrane protein, Wzc, plays a critical role: Mutants lacking Wzc are unable to polymerize high molecular weight capsular polymers. Homologs of Wzc have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria, and therefore the structure and function of this protein are of considerable interest.

NTA-Ni(II)-Nanogold was used to label Wzc with an N-terminal His tag (His6-Wzc); the Nanogold label functioned both as a heavy atom derivative to assist with phasing, and as a high-resolution label to determine the orientation of the complex within the membrane. Comparison of molecular envelopes obtained with and without Nanogold labeling showed that Wzc particles incubated with NTA-Ni(II)-Nanogold were essentially identical to those without Nanogold, but multiple gold densities were easily observed bound to the proteins. With a smaller gold-labeled data set, a second three-dimensional structure at ~22 Å resolution was generated which showed additional bound gold density contained within the volume. Nanogold was found in two locations: on the upper half of the root regions, and at the bottom of the cavity formed by the roots underneath the crown. This second location is not physically connected to the protein envelope, and therefore may represent nonspecific binding or trapped gold particles. However, the presence of Nanogold at either location suggests that the roots contain the N terminus, and hence are in the cytoplasm, while the crown is in the periplasm.

References:

  • Cuthbertson, L.; Mainprize, I. L.; Naismith, J. H., and Whitfield, C.: Pivotal roles of the outer membrane polysaccharide export and polysaccharide copolymerase protein families in export of extracellular polysaccharides in gram-negative bacteria. Microbiol. Mol. Biol. Rev., 73, 155-177 (2009).
  • Collins, R. F.; Beis, K.; Clarke, B. R.; Ford, R. C.; Hulley, M.; Naismith, J. H.; and Whitfield, C.: Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules. J. Biol. Chem., 281, 2144-2150 (2006).

Goes better together:
GoldiBlot™ for His-tag detection

If you are tired of long Western blot protocols, take a look at GoldiBlotTM, our new detection system for His-tagged proteins in Western blots and other immunoblots. GoldiBlotTM is a new type of detection system that provides nanogram-level detection sensitivity within an hour for any protein with a polyhistidine tag. It is based on our Nickel(II)-NTA-gold (nitrilotriacetic acid - Ni(II) - gold) technology, combined with rapid autometallographic amplification, in which metal is selectively deposited onto the bound gold particles. The principle is shown below, together with results obtained for staining His-tagged proteins in a Western blot. Unlike the metal enhancement processes used for organic chromogens, this produces a clean, clear signal without the need for additional reagents or steps.

[GoldiBlot: Result and Principle (46k)]

Left: Western blot detection of His-tagged proteins using GoldiBlotTM HIS Protein Detection kit. Lane M: All Blue protein ladder. Lanes 1-5: His-tagged ATF-1 loaded at 2.5 50 ng (1) 50 ng, (2) 25 ng, (3) 10 ng, (4) 5 ng and (5) 2.5 ng. (6) 100 ng His-tagged YY1. (7) 100 ng His-tagged Src. (8) 50 ng His-tagged Src and bacterial extract with 2,500 ng total E. Coli Protein. (9) bacterial extract with 2,500 ng total E. Coli Protein.

Right: How GoldiBlotTM works: Ni-NTA-Gold binds to His-tagged proteins, and the gold particles are then subjected to autometallographic amplification to render them visible.

Features and advantages include:

  • Detect His-tagged proteins in an hour.
  • Direct visualization of His-tagged proteins in magenta colored bands. No film, autoradiography or phosphorimager are required.
  • More stable than antibodies.
  • Low nanogram-level sensitivity with low background.
  • No antibodies involved.

Applications include:

  • Identify His-tagged proteins rapidly and confidently in cell lysates and extracts.
  • Faster, simpler western blots.
  • Confirm the expression of his-tagged reporter proteins in transfected cells.

Reference:

  • Dubendorff, J.; Cruz, M.; Gonzalez, C.; Hainfeld, J.; Liu, W.: Rapid Detection of His-tagged Proteins on Western Blots Proc. 47th Ann. Mtg., Amer. Soc. Cell Biol., 47; Pres. # 1918., poster # B265 (2007).

More information:

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Nanogold® Labeling Helps Show
Distribution and Function of SAP-1

This month's contribution to the development of pre-embedding Nanogold labeling comes from Sadakata and group, with a paper in the current Genes to Cells. The authors used Nanogold labeling with HQ Silver development to help elucidate the function of Stomach cancer-associated protein tyrosine phosphatase-1 (SAP-1), and its connection with gastrointestinal cancer. SAP-1 (PTPRH) is a receptor-type protein tyrosine phosphatase (RPTP) with a single catalytic domain in its cytoplasmic region and fibronectin type III-like domains in its extracellular region. Until recently, the cellular localization and biological functions of this RPTP have remained unknown. For this study, the authors generated a monoclonal antibody (mAb) against SAP-1 (clone 123), then used secondary immunofluorescence and Nanogold labeling to study the distribution of this protein within the mouse gastrointestinal tract.

 

[Nanogold-Fab' vs. colloidal gold-IgG: resolution (61k)]
Resolution advantage: size comparison of Nanogold-Fab' with conventional 5 nm colloidal gold-IgG probe, showing overall probe size and distance of gold from target. Due to its position at the hinge region, Nanogold is positioned closer to the target upon binding, yet does not hinder or interfere with binding. In addition, its smaller size permits denser labeling and facilitates penetration into tissues and access to hindered antigens.

Immunoblot analysis with this mAb revealed prominent expression of the approximately 250-kDa SAP-1 protein in the intestine, and a low level of the expression in testis. The amount of SAP-1 in the duodenum or jejunum was markedly greater than that in the stomach or colon. Immunohistofluorescence with the mAb to SAP-1 showed that SAP-1 was localized at the apical surface of intestinal epithelial cells, similar to ezrin or alkaline phosphatase, both of which are known to be localized at the microvilli of the intestinal epithelium. SAP-1 immunoreactivity was detected immediately above the prominent staining of F-actin revealed by phalloidin, and this may correspond to the terminal web, at the brush border of intestinal epithelial cells.

For immunoelectron microscopic studies, mice were anesthetized by intraperitoneal (i.p.) injection of sodium pentobarbital at 25 mg/kg of weight, then perfused transcardially with 2% PFA and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were removed and immersed in the same fixative for 1 hour at 4°C, then incubated for 1 hour at 4°C with 1% OsO4 in the same buffer. They were then dehydrated and embedded in Epon. For immuno-EM, tissue samples were fixed by immersion for 2 hours at room temperature in phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA) and 1% glutaraldehyde. Frozen sections of 10 µm thickness were prepared and processed for immunostaining using a mAb to mouse SAP-1, and with Nanogold-labeled Fab' goat anti- rat IgG. Signals were enhanced using HQ Silver Enhancement kit. After silver enhancement was complete, the sections were fixed again with 1% OsO4 and 0.1% potassium ferrocyanide and lastly embedded in Epon. Ultrathin sections (90 nm) were then prepared, stained with uranyl acetate and lead citrate, and examined with a JEM 1010 electron microscope (JEOL).

Analysis of the labeling pattern confirmed that SAP-1 protein localizes to the microvilli of the brush border in gastrointestinal epithelial cells; Mouse SAP-1 mRNA is largely restricted to the gastrointestinal tract. Additional experients found that expression of SAP-1 in mouse intestine is minimal during embryonic development, but increases markedly after birth, coincident with the differentiation of intestinal epithelial cells. SAP-1-deficient mice showed no marked changes in morphology of the intestinal epithelium. However, SAP-1 ablation inhibited tumorigenesis in mice with a heterozygous mutation of the adenomatous polyposis coli gene, resulting in fewer large adenomas (> 2 mm). These results indicate that SAP-1 is a microvillus-specific RPTP that regulates intestinal tumorigenesis.

Reference:

  • Sadakata, H.; Okazawa, H.; Sato, T.; Supriatna, Y.; Ohnishi, H.; Kusakari, S.; Murata, Y.; Ito, T.; Nishiyama, U.; Minegishi, T.; Harada, A., and Matozaki, T.: SAP-1 is a microvillus-specific protein tyrosine phosphatase that modulates intestinal tumorigenesis. Genes Cells, 14, 295-308 (2009).

More information:

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Nanoprobes wins Grants from NIH
to Advance Imaging and Cancer Therapy

Nanoprobes, Incorporated has won three new Phase 1 Small Business Innovation Research (SBIR) grants from the National Cancer Institute and National Institute of Diabetes, Digestive and Kidney Diseases (National Institutes of Health). These grants provide over $500,000 to support Nanoprobes research on the use of gold nanoparticles as imaging and therapeutic agents for the early detection, visualization and therapy of cancer.

The first project, "Improved Renal Diagnoses using Gold Nanoparticle CT Imaging," is the development of gold nanoparticle contrast agents for visualizing kidney structures. Both iodine-based and gadolinium contrast agents are currently subject to concerns and an FDA advisory because of nephrotoxicity concerns. Chronic kidney disease affects up to 4.5% or more of US adults, and the new gold nanoparticle reagents should provide a safe alternative that will provide enhanced visualization for the diagnosis of conditions such as renal arterial stenosis and acute tubular necrosis.

The other two grants will support the investigation of two novel approaches to cancer therapy. In the first, "Nanogold-Enhanced Radiosurgery for Malignant Brain Tumors," gold nanoparticles, delivered preferentially to tumors, will be used to enhance the effect of X-ray radiation therapy on cancer cells. The other, "Infrared & X-ray Nanogold Therapy of Head & Neck Cancers," will investigate the use of gold nanoparticles, targeted to tumors, as intense infrared (IR) absorbers which will be used to enhance infrared phototherapy. Nanoprobes has previously reported promising results from initial experiments in mice, showing that the combination of X-ray therapy and gold nanoparticle delivery provides significant benefits over either approach alone; see:

  • Hainfeld, J. F.; Slatkin, D. N., and Smilowitz, H. M.: The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol., 49, N309-N315 (2004).

More information:

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Other Recent Publications

Reference:

  • Gentry S. T.; Fredericks S. J., and Krchnavek R.: Controlled Particle Growth of Silver Sols through the Use of Hydroquinone as a Selective Reducing Agent. Langmuir, 25, 26132621 (2009).

The precise mechanism of silver enhancement is debatable, but has been of interest since the method was developed. In this month's Langmuir, Gentry and colleagues explore the chemistry of the reduction of silver salts to metal nanocolloids. The authors used hydroquinone as the principal chemical reducing agent to prepare aqueous silver nanocolloids from silver nitrate. It was found that hydroquinone was unable to initiate the particle growth process on its own, but could sustain particle growth in the presence of pre-existing metallic clusters; this unique selectivity that is exploited by silver enhancement. A standard formulation comprised 15 mL of 0.2 mM silver nitrate, 0.2 mM hydroquinone, and 0.2 mM sodium citrate as a colloidal stabilizer diluted to full volume with water. Reactions were buffered using a 0.6 mM 1 : 1 (mol/mol) mixture of monobasic : dibasic potassium phosphate; poly(vinylpyrrolidone) (MW 40,000) was used as a stabilizer. Two different approaches were used to initiate the hydroquinone growth process. In the first, 4.0 10-4 M freshly prepared sodium borohydride was used be used to form 6-8 nm seed particles; the final diameter of the enlarged particles was found to be inversely related to the number of seed particles. In the second, controlled growth was initiated by exposing the samples to UV radiation, relying on the photoreactivity of hydroquinone to start the process; this process required continuous illumination, and because new nuclei were formed throughout the experiment, the size distribution of the enlarged particles was more variable than that of the borohydride-initiated particles. It was also found that the shape of the particles could be modified by adding different stabilizers during the first phase of particle growth: sodium citrate produced nonspherical disk-shaped particles, while poly(vinylpyrrolidone) yielded triangular-plate morphologies directly from solution, without the need for subsequent reformation or template processing.


Reference:

  • Zhang, Y.; Zhang, Y.; Wang, H.; Yan, B.; Shen, G., and Yu, R.: An enzyme immobilization platform for biosensor designs of direct electrochemistry using flower-like ZnO crystals and nano-sized gold particles. J. Electroanal. Chem., 627, 9-14 (2009).

A recent study described in the Journal of Electroanalytical Chemistry confirms the earlier findings of Willner and co-workers that gold nanoparticles can function as electron relays for nanowiring immobilized enzymes. Zhang and co-workers report the development of a novel immobilization platform for fabricating enzyme-based biosensors for the direct electrochemical determination of target molecules, by synergistically using ZnO crystals and nano-sized gold particles. ZnO crystals were synthesized with a flower-like morphology, to provide a larger surface area for anchoring horseradish peroxidase (HRP)-labeled 10 nm colloidal gold particles. The resultant enzyme biosensor was tested for the determination of H2O2 as a test system. Experimental results showed that HRP could be immobilized onto the nanocomposite matrix with at high loading amount, with well-retained bioactivity. Rapid and direct electron transfer could be achieved between the enzyme active sites and the electrode surface, facilitating the direct electroanalysis of H2O2. The enzyme sensor was found to directly determine H2O2 in a concentration range from 1.5 x 10-6 M to 4.5 x 10-4 M, with a detection limit of 7.0 x 10-7 M. High detection reproducibility is anticipated. The ZnOChitosan-colloidal gold enzyme immobilization platform yielded more sensitive hydrogen peroxide detection than a number of alternative platforms, and has a significant potential for the development of direct electrochemical enzyme biosensors.


Reference:

  • Lee, S.; Chon, H.; Lee, M.; Choo, J.; Shin, S. Y.; Lee, Y. H.; Rhyu, I. J.; Son, S. W, and Oh, C. H.: Surface-enhanced Raman scattering imaging of HER2 cancer markers overexpressed in single MCF7 cells using antibody conjugated hollow gold nanospheres. Biosens. Bioelectron., 24, 2260-2263 (2009).

We have previously described the use of colloidal silver particles as substrates for SERS (surface-enhanced Raman scattering), which provides a method for the analysis of bonding within molecules adsorbed to the surface of the particles. In the current Biosensors and Bioelectronics, Lee and group show that hollow gold nanospheres (HGNs) may provide both enhanced performance and biocompatibility that enables their use in living cells. Hollow gold nanospheres were prepared using cobalt nanoparticles, synthesized by reducing CoCl2 with NaBH4 under a nitrogen purging condition, as templates. 0.1MH AuCl4 was added 10 times in 50 µL aliquots: thus, gold atoms were nucleated and grown up to small shells around the cobalt template. When the solution was exposed to ambient conditions by stopping N2 purging, the cobalt dissolved completely, leaving a hollow interior; the color of the solution changed from dark brown to deep blue. The wall thickness could be controlled by changing the concentration of HAuCl4. Using TEM, the diameter of the HGNs and their wall thickness were estimated to be 45±5 nm and 15±3nm respectively. crystal violet (CV) was adsorbed onto the surfaces of the HGNs as a Raman reporter; anti-rabbit IgG was conjugated to the surface of the HGNs using dihydrolipoic acid (DHLA). Two SH terminal groups of DHLA were cleaved and chemically bonded to the HGN surface. Antibody-conjugated hollow gold nanospheres (HGNs) were then used for the SERS imaging of HER2 cancer markers overexpressed in single MCF7 cells. SERS mapping images showed that HGNs have much better homogeneous scattering properties than silver nanoparticles. The results show that HGNs have promise as highly sensitive and homogeneous sensing probes for biological imaging of cancer markers in live cells.

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