Biomolecular Condensates for In Vitro Analysis: Mechanisms and Strategies.

Biomolecular condensates, formed by proteins, nucleic acids, and other macromolecules through liquid-liquid phase separation, enable molecular enrichment and reaction regulation within membraneless microenvironments. As their physical principles have become better understood, condensates are increasingly being incorporated into in vitro analytical systems. Compared with conventional homogeneous assays, condensate-based systems can enhance effective reaction concentrations, regulate molecular partitioning, accelerate reaction kinetics, and facilitate signal generation through enrichment, selective compartmentalization, and spatial confinement. This review discusses the mechanisms by which biomolecular condensates contribute to in vitro analysis, emphasizing component enrichment, selective partitioning, kinetic enhancement, and programmable phase behavior. We then discuss programmable condensate biosensing systems as emerging platform-level frameworks for molecular recognition, signal processing, and multimodal readout. Representative applications are summarized in nucleic acid and protein analysis, enzyme activity regulation, ion and small-molecule detection, and complex sample analysis. Finally, we highlight key challenges for practical implementation, including robust phase-behavior control in complex biological samples, predictive design, reproducibility, standardization, and biological benchmarking.