Advancing Nanofluidic Scattering Microscopy for Label-Free Single-Particle Measurements

Licentiate thesis, 2026

Detection and characterization of biomolecules and biological nanoparticles at the single-molecule and single-particle level is important in fundamental biophysics and applied fields such as therapeutics and diagnostics. However, relying on ensemble methods for their characterization may obscure inherent heterogeneity, including subpopulations or conformations with distinct functional roles. There is also a need for analytical techniques that do not use fluorescent labels or physical tethers that might alter the physicochemical properties of the analyte. In response, label-free optical approaches for single-molecule and single-particle characterization are a growing field where direct characterization of particles in their native state is enabled.

Within the field of label-free single-particle characterization, nanofluidic scattering microscopy (NSM) is a recently developed technique that combines dark-field microscopy with a nanofluidic platform to determine both size and molecular weight of diffusing particles. Detection is enabled by interference between light scattered by the particle and the nanochannel, yielding an optical contrast enabling characterization of proteins ranging from tens to hundreds of kilodaltons in molecular weight.

While NSM has been demonstrated for the characterization of small biomolecules, extending the technique to larger and more complex nanoparticles introduces new challenges. In particular, non-specific adsorption to nanochannel walls has been a major obstacle to NSM characterization. In manuscript I, we overcome this obstacle by introducing a surface passivation strategy based on the formation of a polymer brush layer using the hydrophilic copolymer poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG). The protocol is effective for nanochannel cross-sectional dimensions down to 50 x 50 nm2 and reduces non-specific adsorption by several orders of magnitude. Furthermore, the passivation enables more accurate characterization of proteins and liposomes.

Building on this improvement, manuscript II demonstrates how NSM can be applied to characterize biologically relevant nanoparticles. Specifically, we investigate twelve different lipid nanoparticle formulations and benchmark NSM against established techniques, such as nanoparticle tracking analysis and cryo-transmission electron microscopy, showing good agreement in inferred size distributions. Beyond size distributions, the optical contrast provides a second dimension of particle characterization, offering insights into particle composition and enabling comparison of formulations, as well as assessment of inter-sample heterogeneity.