The detection of residual tetracycline antibiotics (TCs) in the aquatic environment is important because of the irreversible damage of TCs to the ecosystem and human health. Considering the photostability, sensitivity, and accuracy of the detection systems, mesoporous SBA-15 silica material was an ideal support matrix for improving photostability because it can protect the optical properties of fluorescent signals. To achieve ratiometric detection (with sensitivity and accuracy), pyrene and Eu3+ as two fluorescent signals were implemented into SBA-15, and an organic–inorganic hybrid fluorescent nanoprobe Py/EDTA-SBA-15@Eu3+ was constructed. In the presence of TCs, the red emission of Eu3+ in the nanoprobe was sensitized, and the fluorescence resonance energy transfer occurred from pyrene to Eu3+, resulting in the increased emission of Eu3+ at 617 nm and the decreased emission of pyrene at 384 nm. By monitoring the fluorescent intensity changes at 617 and 384 nm (F617/F384), the nanoprobe achieved the ratiometric detection of oxytetracycline (OTC) and tetracycline (TC) in the range of 0.20–80 and 0.40–80 μM, respectively, with detection limits of 0.18 nM for OTC and 5.83 nM for TC. In addition, the practical application of the nanoprobe was validated in tap water and honey samples.
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