Antibody orientation drives sensitivity in SARS-CoV-2 detection using dynamic light scattering biosensors.

The performance of nanoparticle-based biosensors strongly depends on the orientation and density of immobilized antibodies, yet these parameters remain underexplored in platforms based on dynamic light scattering (DLS). This study investigates how the length of molecular crosslinkers influences antibody orientation, surface coverage, and biosensor sensitivity for SARS-CoV-2 detection. The impact of crosslinker spacer length on the analytical performance of 53 nm-gold nanoparticles (AuNPs) functionalized with polyclonal antibodies (pAb) was evaluated using both the spike protein (S Ptn) and intact viral particles as targets. Biosensors were prepared using carboxylic acid-terminated alkanethiol crosslinkers of varying lengths-3-mercaptopropionic acid (MPA), 6-mercaptohexanoic acid (MHA), and 11-mercaptoundecanoic acid (MUA)-and characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and ultraviolet-visible (UV-Vis) spectroscopy. DLS was used to monitor changes in hydrodynamic diameter (ΔDH) upon antigen binding in artificial saliva. Secondary structure analysis and mathematical fitting using the Hill model were employed to evaluate structural effects and binding cooperativity. MUA-functionalized biosensors showed the best performance, with ΔDH shifts up to 115.2 nm for the S Ptn and 77.76 nm for the virus, with respective limits of detection of 2.96 ng/mL and 1.8 × 10 ³ PFU/mL. These improvements were associated with increased antibody packing and favorable end-on orientation. A Hill coefficient of 2.82 in virus detection indicated positively cooperative binding behavior. This study enhances our understanding of antibody-antigen interactions on nanostructured surfaces and guides the development of more effective DLS-based biosensors.