Quantifying Biomolecule Concentration of Phase-Separated Condensates in Optofluidic Chips.

Liquid-liquid phase separation (LLPS) is a fundamental mechanism that drives biomolecular assembly. The concentration of biomolecules within phase-separated condensates is a critical parameter that reflects their physical state, modulates their biological functions, and provides insights into their formation and aging. However, current analytical methods, such as spectroscopy, fluorescence imaging, quantitative phase imaging, and bulk measurements, have limitations in quantifying biomolecule concentrations, involving measurement inaccuracies, sample perturbation, or requirements for large sample volumes. To overcome these challenges, we developed an optofluidic chip that integrates self-aligned micro-optics with a detection well. When LLPS components were mixed at stoichiometric ratios defined by their phase diagrams, condensates naturally formed, fused, and settled in the detection well. Consequently, the reflected light was modulated by the accumulated LLPS condensates on the sensing surface within the chip, enabling in situ, label-free, and reliable measurement of biomolecule concentrations in tiny volumes. Using this optofluidic platform, we quantified biomolecule concentrations for two model condensate systems and one virus-packaging-related condensate system, obtaining values of 168 ± 9 mg/mL for hyaluronic acid-protamine sulfate (HA-PT), 190 ± 13 mg/mL for polyadenylic acid-protamine sulfate (AA-PT), and 235 ± 23 mg/mL for the SARS-CoV-2 RNA SL4 fragment-nucleocapsid protein (SL4-NP). Given these quantitative capabilities, this optofluidic technique holds strong potential for characterizing how diverse LLPS systems evolve with concentration, such as pathological aging of condensates.