Andersson Julia, Lundgren Anders, Olsén Erik, Parkkila Petteri, Midtvedt Daniel, Agnarsson Björn, Höök Fredrik
Department of Physics, Division of Nano and Biophysics, Chalmers University of Technology, Fysikgränd 3, Göteborg 41296, Sweden.
Department of Chemistry & Molecular Biology, University of Gothenburg, Medicinaregatan 7B, Göteborg 41390, Sweden.
J Phys Chem B. 2025 Sep 18;129(37):9506-9516. doi: 10.1021/acs.jpcb.5c04228. Epub 2025 Sep 9.
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.
生物纳米颗粒（NPs）的检测，如病毒和细胞外囊泡（EVs），在医学诊断中起着关键作用。然而，这些颗粒在光学上很微弱，使得在复杂溶液中进行显微镜检测具有挑战性。最近的进展表明，利用偏轴全息显微镜区分金属和介电信号，可以区分金属纳米颗粒和生物纳米颗粒，并对由金属和生物纳米颗粒结合形成的复合物进行定量。在此，该方法被用于研究通过链霉亲和素（StrAv）修饰的金纳米颗粒（StrAv-AuNPs）与含生物素的大单层脂质囊泡（生物素-LUVs）之间的特异性相互作用形成的复合物，生物素-LUVs作为病毒和细胞外囊泡的模拟物。为了在将半径为25 nm的聚乙二醇化金纳米颗粒用四聚体StrAv功能化的过程中使金纳米颗粒的自聚集最小化，使用了0.06%的生物素-聚乙二醇（每个金纳米颗粒约5个生物素），这也有助于确保有效地防止涉及多个脂质囊泡的聚集。虽然在制备StrAv-AuNPs时StrAv与生物素的比例对金纳米颗粒的自聚集没有显著影响，但相对于金纳米颗粒上的生物素-聚乙二醇，需要StrAv过量1000倍才能制备出与生物素-LUVs具有预期反应性的StrAv-AuNPs。通过波导散射显微镜、表面等离子体共振和偏轴全息显微镜的结合，我们证明这可能源于聚乙二醇层内StrAv与生物素之间的缔合速率常数急剧降低。此外，通过使用3 kDa非生物素化聚乙二醇和5 kDa生物素-聚乙二醇的混合物，在StrAv与生物素比例低几个数量级的情况下成功制备了功能性StrAv-AuNPs，使得使用偏轴全息术检测生物素-LUVs的检测限达到亚皮摩尔。
