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

Area of Science:

• Nanotechnology
• Biotechnology
• Analytical Chemistry

Background:

• Nanoparticle biosensor performance relies on antibody immobilization, but orientation and density are often underexplored in dynamic light scattering (DLS) platforms.
• Understanding how crosslinker properties affect antibody presentation is crucial for optimizing DLS-based biosensor sensitivity.

Purpose of the Study:

• To investigate the influence of molecular crosslinker length on antibody orientation, surface coverage, and DLS biosensor sensitivity for SARS-CoV-2 detection.
• To evaluate the analytical performance of gold nanoparticles (AuNPs) functionalized with varying length crosslinkers using spike protein and intact viral particles.

Main Methods:

• Functionalization of 53 nm AuNPs with polyclonal antibodies (pAb) using alkanethiol crosslinkers of different lengths (MPA, MHA, MUA).
• Characterization using ATR-FTIR and UV-Vis spectroscopy; DLS to measure hydrodynamic diameter changes (ΔD_H) upon antigen binding in artificial saliva.
• Secondary structure analysis and Hill model fitting to assess structural effects and binding cooperativity.

Main Results:

• MUA-functionalized biosensors exhibited superior performance, achieving ΔD_H shifts up to 115.2 nm (S Ptn) and 77.76 nm (virus).
• Achieved limits of detection were 2.96 ng/mL for S Ptn and 1.8 × 10³ PFU/mL for the virus.
• Enhanced performance correlated with increased antibody packing and end-on orientation; Hill coefficient of 2.82 indicated positive cooperativity in virus detection.

Conclusions:

• Crosslinker length is a critical factor in optimizing antibody immobilization for DLS biosensors.
• Longer crosslinkers (MUA) promote favorable antibody orientation and density, leading to significantly improved sensitivity and lower detection limits for SARS-CoV-2.
• This research provides insights into antibody-antigen interactions on nanostructured surfaces, guiding the development of more effective DLS biosensors.