ALP-assisted chemical redox cycling amplification for ultrasensitive fluorescent detection of DNA methylation

Affinity assays allow direct detection of DNA methylation events without requiring a special sequence. However, the signal amplification of these methods heavily depends on nanocatalysts and bioenzymes, making them suffer from low sensitivity. In this work, alkaline phosphatase (ALP)-assisted chemical redox cycling was employed to amplify the sensitivity of fluorescence affinity assays for DNA methylation detection using Ru@SiO2@MnO2 nanocomposites as fluorescent probes. In the ALP-assisted chemical redox cycling reaction system, ALP hydrolyzed 2-phosphate-L-ascorbic acid trisodium salt (AAP) to produce AA, which could reduce MnO2 nanosheets to form Mn2+, making the fluorescence recovery of Ru@SiO2 nanoparticles possible. Meanwhile, AA was oxidized to dehydroascorbic acid (DHA), which was re-reduced by tris(2-carboxyethyl) phosphine (TCEP) to trigger a redox cycling reaction. The constantly generated AA could etch large amounts of MnO2 nanosheets and greatly recover Ru@SiO2 fluorescence, amplifying the signal of the fluorescence assay. Employing the proposed ALP-assisted chemical redox cycling signal amplification strategy, a sensitive affinity assay for DNA methylation detection was achieved using ALP encapsulated liposomes that were linked with the 5mC antibody (Ab) to bind with methylated sites. A detection limit down to 2.9 fM was obtained for DNA methylation detection and a DNA methylation level as low as 0.1% could be distinguished, which was superior to conventional affinity assays. Moreover, the affinity assays could detect DNA methylation more specifically and directly, implying their great potential for the analysis of tumor-specific genes in liquid biopsy.