All-Aqueous Phase Segregation Integrated Electrochemical Aptamer Biosensor Enables Picomolar Detection of SARS-CoV-2 Spike Protein

Electrochemical aptamer biosensors (E-ABs) are promising tools for rapid point-of-care (POC) diagnosis which utilize aptamers for the molecular recognition of specific disease biomarkers. However, E-AB sensitivity is typically limited by the binding affinity (K_D) of the aptamer (often in the micromolar-nanomolar range), while biomarker concentrations in biofluids are often at the picomolar level or below. Liquid-liquid phase separation (LLPS) provides a robust framework to concentrate the target biomarker based on affinity-controlled partitioning within an all-aqueous environment. In this study, the integration of LLPS into E-AB is reported to overcome the sensitivity limitation and achieve picomolar Severe acute respiratory syndroome coronavirus 2 spike protein (SARS-CoV-2 S protein) detection. Preferential spike protein partition upon LLPS is verified by microscopic imaging and biochemical assays. The LLPS-integrated E-AB is developed by a peroxidase catalytic reaction to generate electrical signals in the presence of the S protein. The LLPS demonstrates successful protein concentration from synthetic human biofluids and leads to at least 100-fold sensitivity improvement to the picomolar level of the limit of detection (LOD) by altering the volume ratio (VR) of the two segregated phases. This is the first report of an LLPS-embedded E-AB application, advancing the sensitivity of aptamer electrochemical biosensing without a multi-step downstream signal amplification cascade. © 2025 The Author(s). Small published by Wiley-VCH GmbH.