A Phase Separation‐Assisted Pre‐Enrichment Method for Ultrasensitive Respiratory Virus Detection

Abstract Enriching trace biomarkers (e.g., proteins, nucleic acids) is critical for biomedical applications; yet conventional methods often lack versatility, limiting their effectiveness to specific biomarker types. To address this, the phase separation‐assisted pre‐enrichment (PSAP) technology is presented that exploits differential polymer‐polymer partitioning to achieve 47‐fold antigen and 44‐fold RNA enrichment simultaneously. Through systematic optimization of interfacial chemistry, including pH modulation, polymer hydrophilicity, mass fraction, and molecular weights, the protocol is refined to enable direct integration with commercial diagnostics. PSAP‐boosted rapid antigen ests (RATs) detected SARS‐CoV‐2 and Influenza viruses at tenfold and fivefold lower limits, respectively. In clinical validation, 53 clinical specimens (containing PCR undetectable samples as controls) are analyzed. The PSAP method significantly enhanced detection accuracy for both viral antigens and RNA, particularly improving positivity rates in low viral load specimens (27 < Ct < 31) compared to conventional approaches while maintaining specificity in high‐Ct and negative controls. With its universality and tunability, PSAP demonstrates universal applicability across respiratory pathogens and lays the foundation for next‐generation point‐of‐care diagnostics.