The subtle shifts that matter
Biomolecular condensates play an essential role in numerous biological functions such as RNA metabolism, ribosome assembly, signal transduction, and the DNA damage response. Yet, despite their importance, studying condensates remains technically challenging. Standard biochemical and imaging approaches often fail to resolve subtle, concentration-dependent that precede condensate formation or to quantify the degree of compaction involved.
Currently, the most widely used methods to assess liquid-liquid phase separation (LLPS) include fluorescence microscopy, FRAP (fluorescence recovery after photobleaching), turbidity measurements, and optical trapping. While powerful, these techniques are often qualitative, require specialized equipment, and may lack resolution for small-scale complexes or early-stage compaction events.
How can we help?
We built our Fluidity One-M instrument with your challenges in mind. Our proprietary Microfluidic Diffusional Sizing (MDS) technology provides a powerful, quantitative method to assess LLPS and biomolecular condensates.
By directly measuring the hydrodynamic radius (Rh) of biomolecules in solution, MDS detects subtle shifts associated with binding, conformational changes, or compaction, without immobilization or bulk phase separation, making it well-suited to characterize early interaction events and complex formation that precede or occur alongside LLPS.
True size
MDS directly reports the hydrodynamic radius (Rh) of biomolecules, offering precise, quantitative measures of binding, conformational changes, or compaction.
True environment
Experiments run entirely in-solution, free from immobilization or bulk phase separation – so you can study condensates as they naturally occur.
True insights
Detect subtle shifts that other methods miss – gaining a fuller, earlier, and more quantitative picture of condensate formation and dynamics, using just 4µl per sample point.
APPLICATION NOTE
In-solution measurement of RNA compaction and LLPS-driven condensate formation using microfluidic diffusional sizing
Explore how MDS was applied to monitor interactions between three AMPs – P113, Os-C, and Buforin-2 – and structured bacterial RNAs. MDS revealed concentration-dependent RNA compaction and distinguished the relative strength and behaviour of AMP–RNA interactions. This approach provides a quantitative window into biomolecular condensate formation, shedding light on antimicrobial mechanisms that arise from liquid–liquid phase separation.
True size
Direct, in-solution sizing with MDS quantifies peptide–RNA assembly. Tracking Rh (nm) versus polyA cleanly separates AMP behaviours: P113 drives the strongest complex growth, revealing the compaction-to-condensate trajectory without bulk phase separation.
Read more from the original article.
Figures adapted from Sneideris et al., Nat. Commun. 2023, under CC BY 4.0.
True environment
True insights
Figure adapted from Sneideris et al., Nat. Commun. 2023, under CC BY 4.0.
Explore related publications
Quantify collective interactions from single-component readouts
Ausserwöger et al. infer tie-line gradients and energetic dominance from single-component dilute-phase measurements, then use microfluidic diffusional sizing (MDS) as an orthogonal, surface-free, mechanistic validation. MDS tracks the increase in hydrodynamic radius of FUS-EGFP, and similarly TDP43, PGL3 and SOX2, upon increasing 1,6-hexanediol concentration, while EGFP and G3BP1 alone show no change. Together, these MDS readouts pinpoint 1,6-hexanediol as a solvation agent that expands disordered chains and dissolves condensates, tying the compositional energetics directly to a molecular mechanism.
Ausserwöger, Hannes, et al. “Quantifying collective interactions in biomolecular phase separation.” Nature Communications 16 (2025): 7724.
Diagnose mechanisms of condensate modulators
Modulating biomolecular condensates can yield similar phenotypes via different underlying interactions, hindering mechanistic interpretation. Qian et al. extend energy-dominance analysis to classify whether a modulator enhances or suppresses phase separation and whether it acts on the target or its partners, using only dilute-phase concentration measurements. They validate assignments on G3BP1–RNA condensates, where microfluidic diffusional sizing (MDS) of hydrodynamic radius confirms that suramin disrupts protein–RNA binding. The result is a practical, solution-phase workflow for screening and triaging condensate modulators.
Qian, Daoyuan, et al. “Molecular mechanisms of condensate modulation from energy-dominance analysis.” Physical Review Applied 23.6 (2025): 064017.
Map surface patches to nonspecificity and LLPS
Nonspecific interactions can derail antibody developability. Ausserwöger et al. use a rational antibody library and in-solution microfluidics (MDS) to quantify nonspecific binding to ssDNA, then couple this with phase-diagram mapping to link behaviour to surface patch areas. They show that, at physiological salt levels, a hydrophobic/hydrogen-bonding CDR patch drives strong DNA binding. Lowering the ionic strength shifts control to electrostatics: charged patches dominate, promoting cooperative DNA–antibody network assembly and phase separation at low-µM concentrations. The work establishes patch size as a unifying predictor of both nonspecific binding and condensate formation, offering clear levers for sequence design and formulation.
Ausserwöger, Hannes, et al. “Surface patches induce nonspecific binding and phase separation of antibodies.” Proceedings of the National Academy of Sciences 120.15 (2023): e2210332120.
Disassemble PGL-3/RNA condensates
RNA-binding proteins can dismantle condensates through competition for nucleic acids. Lewis et al. reconstitute PGL-3/RNA condensates and, using microfluidic phase-mapping with dilute-phase readouts, show that MEX-5 shifts the phase boundary toward the homogeneous state by sequestering RNA. Domain tests confirm the zinc-finger RNA-binding module is required for disassembly. As an orthogonal, surface-free confirmation, microfluidic diffusional sizing (MDS) tracks hydrodynamic-radius changes during poly-rU titrations to extract binding curves, revealing that MEX-5 binds RNA ~7× more tightly than PGL-3 (≈0.52 µM vs 3.42 µM), while MEX-5ΔZF shows no detectable binding. Together, the phase-diagram/dilute-phase analysis and the MDS readout link mechanism to energetics, quantitatively tying RNA sequestration to a reduced free-energy contribution from PGL-3, and deliver a practical, solution-phase workflow for pinpointing how RNA-binding proteins drive condensate disassembly.
Lewis, Natasha S., et al. “A mechanism for MEX-5-driven disassembly of PGL-3/RNA condensates in vitro.” Proceedings of the National Academy of Sciences 122.20 (2025): e2412218122.