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.
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