Genetic testing plays a crucial role in guiding individualized medication, however, detecting fine structural mutations in genes continues to present significant challenges. This study introduces a dual-signal fluorescence system, termed Fe3O4@Au@PEG@P1+MOF@P2, that integrates magnetic separation of Fe3O4@Au with NH2-MIL-88 (MOF) catalysis. Initially, the specimen (T1/T2) facilitated the formation of a specific complex (Fe3O4@Au@PEG@P1+T1/T2) with Fe3O4@Au@PEG@P1. The subsequent addition of Hoechst-33258 produced a robust fluorescence signal at 460 nm, enabling the identification of mutations in the first gene regions. Following this, MOF@P2 was introduced to activate the catalyst through P2 pairing with T2. The complex Fe3O4@Au@PEG@P1+T1/T2+P2+Hoechst-33258 was subsequently isolated using an external magnetic field. Upon adding OPD, fluorescent DAP was detected at 560 nm, allowing for the identification of mutations in the second gene regions. The research demonstrated that the variation in fluorescence signals increased with a higher number of base substitutions and deletion mutations, with deletion mutations resulting in a notably greater alteration rate compared to substitution mutations. Interestingly, triple base substitution mutations, characterized by lower clustering of non-continuous mutations, produced a more pronounced change in fluorescence signal than did a higher clustering of continuous mutations (codon mutations). This single-step methodology effectively differentiates among the number and types of mutations across multiple gene regions while assessing the degree of mutation clustering. Overall, this technology significantly enhances the current capabilities for detecting fine structural mutations in genes. Furthermore, the approach exhibits high sensitivity in detecting concentrations of T1 and T2 ranging from 10-15 M to 10-9 M, with detection limits of 0.19 fM and 0.24 fM, even in 5 % serum samples.
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