A Real-Time Antifouling Multivalent Aptamer Platform for Wash-Free Electrochemical Detection of Low-Abundance Biomarkers in Human Plasma.

Sensitive and specific detection of low-abundance proteins in complex biofluids is essential for early disease diagnosis and real-time health monitoring. Electrochemical aptamer-based biosensors offer rapid, point-of-care potential, but their clinical translation has been limited by biofouling, matrix variability, and signal instability in samples such as human plasma. Here, we introduce the Real-Time Magnetic Multivalent Aptamer (RT-MagMAp) assay, a one-pot, wash-free electrochemical platform that detects the low-abundance biomarker VEGF₁₆₅ directly in diluted human plasma. The RT-MagMAp system integrates three enabling chemical designs: (i) a hierarchical multivalent aptamer architecture combining bead-immobilized monomeric aptamers with electrode-bound trimeric aptamers to form highly stable electroactive sandwich assemblies; (ii) antifouling zwitterionic polymer coatings that house trimeric aptamers while suppressing nonspecific adsorption; and (iii) a dynamic internal calibration mechanism using nonfunctional mutant aptamers to correct for plasma-dependent variability. Together, these elements enable femtomolar VEGF₁₆₅ detection (32-354 fM, depending on calibration method) and quantitative performance across 124 blinded plasma samples, achieving a Pearson correlation coefficient of 1.00 and a concordance correlation coefficient of 0.996 relative to a commercial ELISA. Together, these results establish RT-MagMAp as a robust, clinically relevant electrochemical platform capable of quantitative, wash-free protein detection directly in complex biological fluids.