TEM Observations of Bio-Conjugated Streptavidin-Gold Nanoparticles
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TEM Observations of Bio-Conjugated Streptavidin-Gold Nanoparticles Ai Leen Koh1,2, and Robert Sinclair3 1 Materials Science and Engineering Department, Stanford University, 416 Escondido Mall, Stanford, CA, 94305 2 Mechanical Engineering Department, Stanford University, 416 Escondido Mall, Stanford, CA, 94305 3 Materials Science and Engineering Department, Stanford University, Stanford, CA, 94305
ABSTRACT Simultaneous visualization of inorganic nanoparticles and their bio-functionalized coating under the TEM can be achieved with negative staining, where electron dense material is introduced onto the specimen to enhance contrast. Using this technique, streptavidin-functionalized gold nanoparticles were negatively stained using phosphotungstic acid (PTA) at pH 7.0 and then observed using the TEM. The proteins appear as light regions/halos surrounding the gold nanoparticles, with widths similar to those of streptavidin protein molecules reported in literature. Experiments were performed to bio-conjugate streptavidin-gold nanoparticles to biotinylated antibodies and biotinylated actin, and then negatively staining the samples with PTA for TEM analyses. In the first experiment, an increase in halo widths in more than 60% of the particles was observed. In the second experiment, the nanoparticles were found to localize around the long actin filaments. Negative staining is useful for studying bio-functionalized nanoparticles as it enables the proteins surrounding the nanoparticles to be imaged and studied using the TEM.
INTRODUCTION Biological applications of nanoparticles usually require that they be functionalized with a biocompatible coating so that they can be tagged to other biomolecules. A common protein used for bio-functionalization is streptavidin, which has a tetrameric structure and molecular weight of 4×15,000Da. Each streptavidin molecule has the ability to bind up to four molecules of biotin with high affinity (dissociation constant Kd =10-15 M). The complexes are also extremely stable over a wide range of temperature and pH. The streptavidin-biotin system forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. The transmission electron microscope (TEM) is a useful tool for studying nanoparticles owing to its sub-nanometer resolution. However, imaging of biomolecules using the TEM poses several challenges. First of all, bio-molecules are composed of light elements which do not scatter electrons sufficiently and hence do not produce contrast. Furthermore, biological specimens are rapidly damaged by the electron beam. It is estimated that as much as 40% of the mass of a macromolecular sample may be lost in a 30 second exposure to the electron beam [1]. To provide high contrast and good preservation of the bio-molecules, negative staining is used. This involves introducing an electron-dense material (stain) onto the TEM grid that holds
the specimen. The stain is mostly excluded by the functionalized nanopart
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