Orthogonal Stimuli-Responsive Y-Shaped DNA Probe for Dual-Antibiotic Photoelectrochemical Detection

Abstract Simultaneously recognizing multiple targets and independently decoding their signals in food is critically demanded for safeguarding public health against antimicrobial resistance. Photoelectrochemical (PEC) sensors, promising for on-site detection, face a fundamental constraint: all targets generate unidimensional current signals at the electrode, rendering multiple target signals prone to overlap and difficult to distinguish. To address this issue, we developed a magnetic-bead-assisted dual-target distinguishable Y-shaped probe (MDDYP) based on orthogonal stimuli-responsive triggers, enabling independent detection of tobramycin (TOB) and tetracycline (TET) on a PEC sensor. Three DNA strands are assembled into a Y-shaped nanostructure bearing hydroquinone (HQ)-encapsulated liposomes, in which the PC-linker and C-rich sequence function as orthogonal stimulus-responsive modules, which are selectively activated after TOB or TET recognition events, respectively. Either UV-induced photocleavage at the PC-linker or acidification-induced i-motif formation at the C-rich sequence drives target-concentration-dependent liposome release into the supernatant, achieving solution-phase signal separation without electrode surface modification. HQ liberated from the lysed liposomes is oxidized at the ITO/ZIF-67/Fe3O4/Fe-COFBTC photoanode, altering the electron transfer pathway and reversing the photocurrent polarity as the quantitative readout. The platform demonstrates a wide linear range of 1 fM–100 nM for TOB and 10 fM–100 nM for TET, with detection limits of 0.62 and 2.27 fM, respectively. Featuring simple operation, rapid response, and dual-target detection, this work offers a promising strategy with potential for future integration into on-site platforms for multiplexed detection of antibiotic residues.