Compartmentalization and Aggregation of Biomolecular Condensates in Crowded Hydrogels for Enhanced Nucleic Acid Diagnosis

Membrane-less compartments, such as biomolecular condensates, play a crucial role in cellular organization by enhancing enzymatic reaction efficiency through compartmentalization and crowding. This has considerable potential for improving in vitro diagnostics. However, the practical biomedical applications of intracellular microenvironments for in vitro diagnostics remain in their infancy. Herein, inspired by membrane-less compartments from biomolecular condensates, a crowded hydrogel-based approach to mimic the intracellular microenvironment through the biocompatible porous structure of hydrogels, thereby promoting the compartmentalization and aggregation of enzymatic reactions to achieve highly sensitive in vitro nucleic acid diagnosis, is proposed. This hydrogel-based system demonstrates significantly enhanced polymerase chain reaction amplification efficiency through enhanced primer-template binding and enzyme activity-mediated single-stranded DNA extension. This enhancement primarily arises from improved molecular interactions driven by excluded-volume effects in crowded environments as well as hydrogel compartmentalization. With high enzymatic efficiency, a two-orders-of-magnitude lower detection limit for pathogens is achieved. These findings suggest that crowded hydrogels have the potential to bridge the gap between the intracellular environment and in vitro applications, offering a novel strategy for advanced molecular diagnostics.