Temperature-Responsive Nanocarrier-Regulated Alternative Release of "Cargos" for a Multiplex Photoelectrochemical Bioassay of Antibiotic-Resistant Genes.

A smart temperature stimuli-driven multiplex photoelectrochemical (PEC) assay was constructed for antibiotic resistance genes (ARGs) detection, where the stimuli-responsive gatekeeping by regulating the alternative release of "cargo" allowed for the simultaneous detection of multiple tetracycline resistance gene, using tetA (T_DNA1) and tetC (T_DNA2) as the model. Dual temperature-responsive nanoassemblies were embedded in the PEC bioassay as signal DNA tages: one thermoresponsive polymer (poly(<i>N</i>-isopropylacrylamide), PNIPAM)-capped mesoporous silica nanoparticles (MSN) with loading the "cargo" of HgO nanoparticles as signal DNA1 tags (S_DNA1-PNIPAM@MSN@HgONPs) and the other antimony tartrate (SbT)-anchored silica nanospheres as signal DNA2 tags (S_DNA2-SbT@SiO₂NSs). At 20 °C, below the lower critical solution temperature (LCST) of PNIPAM, the "gatekeeper" PNIPAM in S_DNA1-PNIPAM@MSN@HgONPs was in an ON state, igniting Hg²⁺ release through the pore of SiO₂. While at above LCST (40 °C), it was in an OFF state. Likewise, the thermo-dependent dissociation of SbT endowed the grafted S_DNA2 tags switching from the OFF (at 20 °C) to ON state (at 40 °C), igniting SbO⁺ release. The released Hg²⁺ and SbO⁺ triggered the amplified photocurrents due to the structure evolution of the photoactive layer into HgS/ZnS or Sb₂S₃/ZnS heterostructure, thus achieving sensitive detection of multiple ARGs: tetA, tetC, tetG, tetM, tetO, tetZ, tetX, and tetW. Combined with heat map analysis, rapid screening of the ARGs profiles in 12 samples could be realized. This bioassay is simple and accessible for multiple genes analysis with the detection limit down to 0.50 nM. And it was successfully applied for measuring tetracycline ARGs in real sludge samples.