ArticleThe journal of physical chemistry. B2025
Quantitative Detection of Biological Nanoparticles Using Twilight Off-Axis Holographic Microscopy: Insights on Complex Formation between PEGylated Gold Nanoparticles and Lipid Vesicles.
Article in The journal of physical chemistry. B, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
The detection of biological nanoparticles (NPs), such as viruses and extracellular vesicles (EVs), plays a critical role in medical diagnostics. However, these particles are optically faint, making microscopic detection in complex solutions challenging. Recent advancements have demonstrated that distinguishing between metallic and dielectric signals with twilight off-axis holographic microscopy makes it possible to differentiate between metal and biological NPs and to quantify complexes formed from metal and biological NPs binding together. Here, this method is employed to investigate complex formation through specific interactions between streptavidin (StrAv)-modified gold NPs (StrAv-AuNPs) and large biotin-containing unilamellar lipid vesicles (biotin-LUVs), serving as virus and EV mimics. To minimize AuNP self-aggregation during functionalization of PEGylated 25 nm radius AuNPs with tetrameric StrAv, 0.06% biotin-PEG (∼5 biotin per AuNP) was used, which also serves to ensure that aggregation involving multiple LUVs is effectively prevented. While the StrAv-biotin ratio did not significantly affect AuNP self-aggregation upon fabrication of StrAv-AuNPs, a 1000-fold StrAv excess with respect to biotin-PEG on the AuNPs was required to fabricate StrAv-AuNPs with the anticipated reactivity with biotin-LUVs. Through a combination of waveguide scattering microscopy, surface plasmon resonance, and twilight off-axis holographic microscopy, we demonstrate that this likely stems from a dramatic reduction in the association rate constant between StrAv and biotin within the PEG layer. Furthermore, by using a mixture of 3 kDa nonbiotinylated PEG and 5 kDa biotin-PEG, functional StrAv-AuNPs were successfully fabricated at an orders of magnitude lower StrAv-to-biotin ratio, enabling a sub-pM limit of detection of biotin-LUVs using off-axis holography.
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