Ultrasensitive Biosensing Platform Based on Hydroxylamine-Enhanced Copper-Mediated Fenton-Like Reaction: Application to Cu(II) and Nucleic Acid Detection

The burgeoning field of reactive oxygen species (ROS)-based biosensing holds significant promise for advancements in disease diagnosis and therapeutic monitoring. Herein, we describe the innovative development of a hydroxylamine (HA)-mediated copper-based Fenton-like reaction (FLR) system. A key advantage of this approach lies in its ability to function optimally at neutral pH, a critical departure from traditional iron-catalyzed Fenton reactions that are hindered by metal ion hydrolysis. By incorporating HA as a cyclic reductant, we engineered an efficient amplification mechanism [Cu(II)→HA→Cu(I)→H₂O₂→^•OH], amplifying hydroxyl radical (^•OH) generation by 5.6-fold. This robust amplification enabled the development of a sensitive copper ion detection assay exhibiting a wide linear range (0.05-100 nM) and an exceptionally low limit of detection (LOD) of 0.01 nM, which was subsequently validated in biological matrices such as human hair. Furthermore, magnetically carboxylated copper-loaded iron oxide nanoparticles (Cu/Fe₃O₄-COOH) were synthesized for the first time as highly efficient metal carriers, enabling the ultrasensitive detection of nucleic acids. Through a sophisticated assay design incorporating DNA hybridization probes labeled with these nanoparticles and efficient Fe³⁺ masking by F^-, we achieved ultrasensitive and sequence-specific detection of hepatitis B virus (HBV) DNA. The assay displayed a linear response from 0.1 to 10 nM with an LOD of 0.06 nM, and demonstrated excellent performance in clinical serum samples with spiked recoveries ranging from 97.1 to 108.0%. This research not only introduces a simple strategy for metal ion sensing but also paves the way for the rational design of ultrasensitive nucleic acid biosensors. The demonstrated utility of Fe₃O₄-COOH as a functionalizable nanocarrier opens avenues for its application in diverse biosensing platforms and other fields requiring advanced nanomaterials.