Abstract

Fluorescent aptasensors are promising molecular diagnostic platforms due to their high specificity, stability, and rapid response; however, their sensitivity for trace-level detection is often limited by the low recognition efficiency of aptamers and suboptimal photophysical properties of fluorescent reporters. Herein, we report an enhanced fluorescent aptasensor via tetrahedral DNA framework-mediated multivalent aptamer network (MAN) and high-quantum-yield carbon quantum dots (CQDs). The MAN is constructed by grafting multiple aptamers onto a reverse tetrahedral DNA (RTDNA) scaffold and magnetic nanoparticles, with three vertices of RTDNA extending outward as duplexed aptamer arms. The resulting radially oriented and flexible aptamer architecture improves target accessibility and enables multivalent binding, yielding an approximately 3-fold increase in affinity compared to a monovalent aptamer. Meanwhile, CQDs with a quantum yield of 43.85%, engineered via a multiobjective optimization strategy, generate a sensitive “fluorescence-on” signal through MAN-regulated DNA polymerization. This aptasensor exhibits a linear range of 10 2 –10 6 pg/mL and a detection limit of 23.96 pg/mL for aflatoxin B1, and performs reliably in complex food matrices, demonstrating its applicability for trace-level detection.