Frederic R. Furuya

Large Covalently Linked Fluorescent and Gold Nanoparticle Immunoprobes

FluoroNanogold™ probes containing both a fluorescent label and the Nanogold® cluster covalently linked to a targeting molecule have been successfully used for correlative fluorescence and electron microscopy (EM) [1]. However, Nanogold is small (1.4 nm) and requires silver or gold enhancement for visualization [2]. This produces much greater size variability than is found in larger colloidal gold preparations. Silver enhancement may lead to non-specific background; and silver can be chemically etched by osmium tetroxide (OsO4) fixation leading to signal loss [2]. We have prepared covalently linked combined fluorescent and larger gold nanoparticle probes for correlative microscopy that can be directly visualized by EM without the need for autometallographic enhancement. Gold nanoparticles, 2 nm to 40 nm in diameter, were stabilized and functionalized using a self-assembling coating comprising a hydrophobic chelating thiol domain to seal the gold surface from aqueous media, and hydrophilic terminal functional groups (polyethylene glycol, PEG) that biocompatibilize the gold nanoparticles and enable site-specific covalent probe conjugation. This provides high stability and a wide choice of surface properties and conjugation reactions. 5 nm gold nanoparticles were prepared by direct reduction of aqueous solution of gold (III) salt with sodium borohydride in the presence of a mixture of solubilizing non-functional and protected amine-functionalized thiols, followed by purification using sucrose density gradient (10–30%) ultracentrifugation (Ti-41 rotor, 26,000 rpm, 45 min, 5 °C; the intense red band that travels ~ 4 cm from the top contains 5 nm gold). Amine-functionalized gold particles were prepared by deprotection in methanolic hydrochloric acid. Specific reactivity was demonstrated by reaction with commercial N-hydroxy succinimido-(NHS) Cy-5.5 fluorescent dye. This conjugate was further purified by Superose-12 size exclusion chromatography (Figure 1). Unstabilized particles of this size are precipitated by 0.1 M sodium chloride, but the coated particles remained dispersed and suspended, even in 1.0 M sodium chloride solution. They were repeatedly centrifuged and completely resuspended without leaving any solid precipitate behind; identical treatment of conventional protein or macromolecule-stabilized colloidal gold conjugates always results in loss of a fraction as an insoluble pellet that cannot be resuspended. Figure 1 UV-visible, fluorescence (Exλ 660 nm) spectra, and TEM (a, b, and c) of fluorescent 5 nm gold-Cy-5.5 conjugate purified by density gradient ultra-centrifugation (d) followed by size exclusion chromatography (vide supra). Combined 5 nm gold-secondary antibody-Alexa Fluor 594 conjugates were prepared by covalently linking NHS- and Maleimido- activated 5 nm gold particles to the secondary antibody molecule, IgG and F(ab′) respectively. NHS-modified Alexa Fluor 594 dye was then reacted with the conjugate. The product was purified using sucrose density gradient ultracentrifugation followed by gel filtration as above. The anti-rabbit-F(ab′) conjugate was used as a secondary probe against a polyclonal rabbit anti-red blood cell antibody to label sheep red blood cells in suspension and the labeling was observed by fluorescence microscopy using Nikon G-2A filter set (Figure 2). The anti-mouse-IgG conjugate was used as a secondary probe with mouse anti-human AE1/AE3 primary that produced clear staining of cytokeratin in human tonsil tissue (Figure 3). The relative quantum yields for the fluorescent gold conjugates were 22–35% of the corresponding commercial dye labeled secondary antibodies. Figure 2 Bright field (A) and fluorescence images (B) of sheep red blood cells labeled with 5 nm gold anti-rabbit-F(ab′)-Alexa Fluor 594; C and D are images for control cells in which the primary rabbit anti-sheep red blood cell antibody was excluded. Figure 3 Fluorescence (A), bright field (B), and bright field image following silver development (C) of cytokeratin stained tonsil tissue with mouse anti-human AE1/AE3 primary and 5 nm gold anti-mouse-IgG-Alexa Fluor 594 secondary antibody; D is corresponding ...

Gold Nanoparticle Photoaffinity Labels for Electron Microscopy

The conventional methods for steric stabilization of transition metal and semiconductor nanoparticles (NPs) involve the use of organic or natural polymers, surfactants, lipid bilayers and silica coatings. These methods significantly increase overall size of the NPs [1], and that could be problematic for some applications because of dielectric nature of the coating versus the conducting or semiconducting properties of the metal core may affect the properties and performance of the stabilized NPs in the desired end-application. To reduce overall size of the stabilized NPs we have developed a selfassembling coating comprising a hydrophobic metal chelating domain that seals the metal surface from aqueous buffers and a variable length hydrophilic terminal groups that stabilize and biocompatibilize the NPs (Figure 1). The terminal groups also provide means to further functionalize and cross-link NPs to specific groups when a small proportion (10-30%) of activatable terminal groups, e.g., -NH2 or -COOH, are included on the NP surface. We have used this strategy to develop metal NP labels for microscopic localization and imaging [2,3]. We now report gold nanoparticle (AuNP) photo-affinity labels (PALs) prepared following a similar strategy. PALs enable direct verification of the spatial proximity of macromolecular components of proteins that are not amenable to crystallography or NMR [4]; the smaller-sized heavy metal NP labels will enable unambiguous localization macromolecular components by electron microscopy and tomography at higher resolution.

Gold Nanoparticle Technology to Address Variability in EM Labeling

Conventionally, colloidal gold labeled antibodies have been the method of choice for electron microscopic labeling, but suffer from limitations. Reliable conjugation protocols exist only for antibodies and a few proteins and require additional macromolecules for stabilization: these yield large probes that penetrate slowly and may only label a small fraction of closely spaced targets. When used as secondary probes especially, the “radius of uncertainty,” or distance from the binding site to the gold label, may be as large as 15-25 nm or more, prohibiting the differentiation of discrete targets in small structures such as synapses. In addition, multiplexing is traditionally achieved by the use of labels of different sizes. Because each preparation of colloidal gold contains a range of sizes, multiplexing by size is generally limited to two or three targets. Furthermore, differences in probe size, which limits antigen access and labeling density, can mean that labeling results may not reflect the relative abundance of the different targets.

Combined Texas Red and 1.8 nm FluoroNanohold™ for Multimodal Imaging

* Nanoprobes, Incorporated, 95 Horseblock Road, Unit 1, Yaphank, NY 11980 ** Dept. of Physics and Astronomy, Stony Brook University, Stony Brook, NY 11794 *** Dept. of Materials Science and Engineering, Stony Brook University, Stony Brook, NY 11794 **** Dept. of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794 ¶ Present address: Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025 † Present address: Dept. of Physics and Astronomy, Northwestern University, Evanston, IL 60208 and Argonne National Laboratory, Argonne, IL 640439

Nanogold Labels, Covalent Gold, and Enzyme Metallography as Components of Nanodevices

Covalently linkable gold nanoparticles provide both highly sensitive and precise localization and detection of biological targets, and components that may be integrated into nanoscale devices with molecular precision to impart a variety of useful properties. The applications of the 1.4 nm Nanogold particle to biological staining and detection are described, and its applications to nanostructured materials and nanodevices are reviewed. In addition, a new method for depositing metal from solution using targeted enzyme probes, EnzMet (enzyme metallography) allows highly sensitive and specific detection of genetic and protein targets in cells and tissues, and has been used to develop an improved test for HER2 gene amplification in breast cancer. EnzMet also provides a novel method for immunoelectron microscopic staining, a reagent for highly sensitive blotting, and a method for target detection on conductive array biochips.

High Z Metal Carbonyls for Imaging and Microspectroscopy

The power of microscopic and crystallographic techniques in structure-function analysis of multi-protein complexes can be greatly enhanced by the use of high Z or heavy atom labels introduced at specific sites. Heavy metal cluster complex labels that can be derivatized for site-specific covalent attachment to macromolecules have clear advantages over heavy metal salts. We have commercialized combined fluorescent and heavy metal probes that enable collection of complimentary sets of data, from fluorescence and electron microscopy, for correlative microscopic studies of biological targets at different spatial resolutions. We herein report combined heavy metal probes that will enable detection and spatially resolved chemical analysis of biological samples.