Liquid-Liquid Phase Separation for Analyte Enrichment in Coacervates for Ultra-Sensitive Biosensing

Efficient isolation and concentration of biomolecules are foundational for rapid diagnostics. Traditional methods, often reliant on solid surfaces, necessitate complex and labor-intensive procedures. Liquid-liquid phase separation (LLPS) systems emerged as promising due to their ability to extract biomolecules via the partition effect. Inspired by the biomolecular compartmentalization observed in cellular organelles, we form coacervates via associative LLPS for concentrating a diverse range of biomolecules, significantly enhancing sensitivity and lowering detection limits of biomolecules by condensing biomolecules in compartments. We demonstrate that under optimal conditions these coacervates can achieve an enrichment factor of up to 22.8 for DNA, surpassing the efficiency of segregative LLPS. This results in significantly enhanced sensitivity for DNA detection. Through fluorescence microscopy, microplate readers, and flow cytometry, we establish that coacervate droplets are approximately 21 times more effective in reducing the limit of detection (LOD) for DNA compared to segregative LLPS droplets. The superior performance of coacervates is attributed to non-covalent interactions between DNA and molecules within coacervates, enabling ultra-sensitive quantification without the need for complex instrumentation or signal amplification. By creating distinct compartments for biomolecule condensation, our approach not only simplifies the detection process but also significantly lowers detection thresholds, paving the way for more accessible and rapid diagnostic methods.