A novel thymine- and guanine-rich oligonucleotide (ODN-7) was engineered explicitly for the detection of Hg2+ and Pb2+ by a single intercalated dye 4',6-diamidinyl-2-phenylindole (DAPI). Upon the introduction of Hg2+, a rapid formation of T-Hg2+-T base pairs takes place, triggering the assembly of a split G-quadruplex structure, resulting in a strong fluorescence signal due to DAPI intercalating into the T-Hg2+-T mismatch. The introduction of Pb2+ initiates an interaction with the split G-quadruplex, causing a significant conformational change in its structure. Consequently, the altered split G-quadruplex structure fails to facilitate the insertion of DAPI into the T-Hg2+-T complexes, leading to fluorescence quenching. This strategy offers a straightforward means of detecting Hg2+ and Pb2+. Leveraging the split G-quadruplex, the ODN-7 sensor enables the detection limits (3σ) for Hg2+ and Pb2+ to reach an impressive low of 0.39 nM and 4.98 nM, respectively. It exhibited a favorable linear range of 0.39-900 nM for Hg2+ detection (R2=0.9993) and 4.98 nM-5 μM for Pb2+ determination (R2=0.9953), respectively. Furthermore, the proposed sensor had excellent selectivity for detecting Hg2+ and Pb2+. It was used in milk samples containing mixed Hg2+ and Pb2+ solutions, yielding recovery rates of 99.3%~103.8% for Hg2+ detection and 100.1%~104.1% for Pb2+ detection.

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