Advancing evanescent light scattering microscopy for single-particle characterization of gene delivery nanoparticles

Precise and reproducible single-nanoparticle characterization is essential for advancing gene therapy, where viral vectors encapsulating therapeutic genes must be evaluated under formulation-relevant conditions. We present a label-free optical platform based on dark-field total internal reflection microscopy (DF-TIRM), developed to image and quantitatively characterize nanoparticles near a passivated glass–water interface using evanescent illumination. The system employs a prism-based total internal reflection (TIR) geometry to selectively illuminate near-surface particles, enabling high-contrast scattering detection while rejecting incident light. Using polystyrene nanospheres with a diameter of 100 nm as a well-defined model system, we systematically investigate how solution conditions and glass surface functionalization influence undesirable nonspecific particle sticking over a range of NaCl concentrations. On bare glass, polystyrene beads can be imaged without significant sticking only at very low NaCl concentration (≤ 1 mmol/L NaCl); above 10 mmol/L, immobilized particles rapidly accumulate and overcrowd the surface. Supported lipid bilayers markedly reduce sticking at intermediate salt (20 mmol/L to 50 mmol/L) but still accumulate immobilized beads at 100 mmol/L to150 mmol/L. Polyethylene glycol (PEG) treated (PEGylated) glass, in our implementation, performs poorly without surfactant, exhibiting even higher sticking than bare glass at 20 mmol/L. Finally, we show that adding a volume fraction of 0.1 % Polysorbate 20 to 150 mmol/L NaCl dramatically reduces sticking on both bilayer and PEG-coated surfaces. These results provide practical guidelines for selecting surface and buffer conditions that preserve suspended particles within the evanescent field and lay the groundwork for future DF-TIRM measurements on viral gene-delivery vectors.