INTRODUCTION
The pursuit of experimental methods for high-resolution characterization of biomolecular properties continues to drive innovation in analytical technology development. Size and shape in particular are critical stereometric discriminators between molecular species and states in solution. We describe a microchip-based method that leverages the combined advantages of random thermal motion and size-dependent confinement effects at the nanoscale to achieve high measurement sensitivity to molecular conformation under native conditions in the solution phase.
RATIONALE
This approach visualizes the motion of fluorescently labeled molecules within fluid-filled slits using widefield microscopy. Shallow slits constrain molecular movement vertically, whereas periodic indentations act as entropic traps, increasing the time molecules spend in deeper regions. This effect is size-dependent: Larger molecules remain trapped longer. By engineering the number of accessible states—translational, configurational, and conformational—entropy can be harnessed at the nanoscale to amplify the influence of molecular size and shape on escape times. Precise measurement of these escape times furnishes insight into both the hydrodynamic radius and the diameter of the smallest bounding sphere enclosing the molecule, establishing a crucial link between solution-phase three-dimensional (3D) conformations and molecular models.
RESULTS
The technique offers a broad, customizable dynamic range for molecular weight measurements, spanning from 500 Da to at least 500 kDa. It can distinguish differences as small as two carbon atoms in small molecules and process complex samples. The single-molecule detection fosters exceptional sensitivity, currently detecting molecular concentrations as low as 10 fM. The method’s speed and precision support quantification of intermolecular interaction strengths across more than six orders of magnitude in affinity constant and enable real-time monitoring of reaction kinetics. By tracking individual molecules, the approach also resolves molecular-state heterogeneity at the highest resolution. Lastly, we demonstrate the diagnostic potential of the technique by leveraging ligand-induced conformational changes in the insulin receptor to sense insulin levels in serum.
CONCLUSION
Escape-time stereometry (ETs) delivers (i) quantitative insight into molecular 3D structure, (ii) thermodynamic and kinetic data on interactions, and (iii) a high-speed, high-sensitivity detection platform for diagnostics, addressing a long-standing integration challenge in molecular measurement technology. ETs is likely to be particularly effective for detecting multimeric complexes formed through weak interactions, which are often difficult to identify with other methods. By enabling high-throughput, solution-phase conformation mapping to molecular models, ETs could support machine learning approaches for 3D structure prediction, validation, and inference. This capability is particularly valuable for studying complex structural problems, such as disordered proteins and RNA, and for detecting and characterizing rare molecular states.
