Antibiotic resistance, encoded via particular genes, has become a major global health threat and substantial burden on healthcare. Hence, the facile, low-cost, and precise detection of antibiotic resistance genes (ARGs) is crucial in the realm of human health and safety, especially multiplex sensing assays. Here, a smart pH-regulated switchable photoelectrochemical (PEC) bioassay has been created for ultrasensitive detection of two typical subtypes of penicillin resistance genes bla_-CTX-M-1 (target 1, labeled as T_DNA1) and bla_-TEM (target 2, labeled as T_DNA2), whereby pH-responsive antimony tartrate (SbT) complex-grafted silica nanospheres are ingeniously adopted as signal DNA1 tags (labeled as S_DNA1-SbT@SiO₂NSs). The operations of the PEC bioassay depend on the switchable dissociation of the pH-responsive S_DNA1-SbT@SiO₂NSs complex under the external pH stimuli, thus initiating the pH-regulated release of ions pre-embedded in sandwich-type DNA nanoassemblies. At acidic conditions, the dissociation of S_DNA1 tags (ON state) triggers the release of the embedded SbO⁺. Under alkaline conditions, the dissociation of S_DNA1 tags is inhibited (OFF state). The detection of T_DNA2 was achieved via DNA hybridization-triggered metal ion release. The unwinding of the introduced hairpin T-Hg²⁺-T fragment, hybridized with the second anchored signal DNA (S_DNA2), ignites the release of Hg²⁺. The released SbO⁺ or Hg²⁺ ions would trigger the formation of Sb₂S₃/ZnS or HgS/ZnS heterostructure through ion-exchange with the photosensitive ZnS layer, giving rise to the amplified photocurrents and eventually realizing the ultrasensitive detection of penicillin resistance genes subtypes, bla_-CTX-M-1 and bla_-TEM. The as-fabricated pH-regulated PEC bioassay, smartly integrating the pH-responsive intelligent unit as S_DNA tags, pH-regulated release of embedded ions, and the subsequent ion-exchange-based signal amplification strategy, exhibits high sensitivity, specificity, low-cost, and ease of use for multiplex detection of ARGs. It can be successfully used for measuring bla_-CTX-M-1 and bla_-TEM in real E. coli plasmids, demonstrating great promise for developing a new class of genetic point-of-care devices.

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