Ratiometric fluorometric assay triggered by alkaline phosphatase: Proof-of-concept toward a split-type biosensing...

Herein, a sensitive ratiometric and split-type fluorescent sensing platform has been constructed for DNA detection based on one signal precursor and two fluorescent signal indicators. In this assay, o-phenylenediamine (OPD) was selected as the signal precursor. On one hand, Cu²⁺ can oxidize OPD to produce 2, 3-diaminophenazine (DAP), which with an emission peak at 555 nm. On the other hand, ascorbic acid (AA) could react with Cu²⁺ to generate dehydroascorbic acid (DHAA), which could further react with OPD to form 3-(1, 2-dihydroxy ethyl)furo[3, 4-b]quinoxalin-1 (3H)-on (DFQ) with a strong emission peak at 420 nm. As a result, the formation of DAP was inhibited, and leading to the decrease of fluorescence intensity at 555 nm. Alkaline phosphatase (ALP) could catalyze the substrate l-ascorbic acid-2-phosphate (AA2P) to produce AA in situ. Inspired by the successful use of ALP as a biocatalytic marker in bioassay, a split-type ratiometric fluorescent assay has been designed for DNA detection by using H1N1 DNA as the target model. It was realized for ratiometric fluorescent determination of H1N1 in a linear ranging from 50 pM to 1.5 nM with a limit of detection of 10 pM. The novel strategy could reduce the mutual interferences between the biomolecular recognition system and the fluorescence signal conversion system, which improving the accuracy of detection and effectively reducing the background signal. Furthermore, the strategy provided a promising platform for biomarkers detection in the fields of ratiometric fluorescent biosensors and bioanalysis.