# Label-free mass and size characterization of few-kDa biomolecules by hierarchical vision transformer augmented nanofluidic scattering microscopy

## TL;DR

A new method uses nanofluidic scattering microscopy and AI to measure the mass and size of small biomolecules like insulin without labels.

## Contribution

The method extends nanofluidic scattering microscopy to characterize molecules as small as 6 kDa using a hierarchical vision transformer.

## Key findings

- The method can measure molecules as small as 6 kDa and 1.5 nm in hydrodynamic radius.
- Using a hierarchical vision transformer improves accuracy close to the theoretical Cramér–Rao Lower Bound.
- The approach is effective for biologically relevant molecules like cytokines and peptide hormones.

## Abstract

Nanofluidic scattering microscopy characterizes single molecules in subwavelength nanofluidic channels label-free, using the interference of visible light scattered by the molecule and nanochannel. It determines a molecule’s hydrodynamic radius by tracking its diffusion trajectory and its molecular weight by analyzing its scattering intensity along that trajectory. However, using standard analysis algorithms, it is limited to characterization of proteins larger than  ≈ 60 kDa. Here, we push this limit by one order of magnitude to below  ≈ 6 kDa molecular weight and  ≈ 1.5 nm hydrodynamic radius — as we exemplify on the peptide hormone insulin — by using ultrasmall nanofluidic channels and by analyzing the data with a hierarchical vision transformer. When we benchmark this approach against the theoretical limit set by the Cramér–Rao Lower Bound, we find that it can be approached with sufficiently long molecular trajectories. This enables quantitative label-free single-molecule microscopy for biologically relevant families of sub-10-kDa molecules, such as cytokines, chemokines and peptide hormones.

The study shows that label-free nanofluidic scattering microscopy combined with an AI vision transformer can measure the mass and size of single biomolecules down to 6 kDa (1.5 nm), as demonstrated on the example of insulin.