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

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.