ABSTRACT 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 VEGF 165 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 VEGF 165 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.
Sen et al. (Mon,) studied this question.