Enhanced Diagnostic Tools
Passive adsorption of a protein molecule onto silver nanoparticles is mediated by the electrostatic and hydrophobic interactions between the protein molecule and the surface layer of the colloidal silver. This process is maximally achieved at a pH close to the pI of the protein to be conjugated. Another important parameter is the amount of protein loading. If surface-adsorbed protein is not sufficient, aggregation occurs upon addition of electrolytes present in standard buffers. Therefore, a titration is required to determine the protein concentration for complete saturation and shielding of the silver nanoparticle surface.
However, some proteins may undergo disturbances to the tertiary structure upon passive adsorption, which may impair their affinity and/or specificity in molecular binding applications. To better preserve the activity of the conjugated protein an alternative approach can be used, i.e., covalently coupling protein molecules onto silver nanoparticles functionalized with carboxyl groups via a PEG-linker. The PEG-linker allows for more flexibility and also better accessibility of the conjugated protein to its antigen/substrate due to the inherent mobility of the PEG-linker and the increased distance from the gold surface. Further, the shielding polyethylene glycol (PEG) layer generally also result in conjugates with superior stability and less non-specific binding. Conjugation to these types of functionalized particles can be performed using straightforward carbodiimide (EDC/NHS) coupling chemistry.
Note: The amount of protein needed to saturate the silver colloid can also be determined and verified through agarose gel-electroporesis. Binding of protein to the silver nanoparticle surface changes the overall particle charge and size both of which will affect the migration pattern in the agarose gel.
Figure 1.Validation of functionality (biotin binding) of a streptavidin silver nanoparticle conjugate using an immuno-dot blot assay.
Table I. Recommended centrifugation speeds for silver nanoparticles of different sizes. Note that centrifugation times and speeds are based on a 1 ml sample volume in 1.5 ml microcentrifuge tubes.
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