Magnetically controlled 2D nano-DNA fluorescent biosensor for selective and sensitive detection of alkaline phosphatase activity

Abstract

In this work, a novel magnetically controlled two-dimensional nano-DNA fluorescence sensor for sensitive and selective detection of alkaline phosphatase (ALP) activity has been developed by combining the λ exonuclease (λ exo) cleavage reaction and a δ-FeOOH nanosheet-based platform. A 5ʹ-phosphoryl-modified double-stranded DNA probe (5ʹ-P-dsDNA) was designed as a substrate for ALP, and a 5ʹ-dye-labeled single-stranded DNA/δ-FeOOH system was designed as a detection unit. In the absence of ALP, 5ʹ-P-dsDNA was degraded by λ exo to yield mononucleotides. Thus, the fluorescence of the detection unit was quenched. However, in the presence of ALP, the 5ʹ-phosphate functional group of 5ʹ-P-dsDNA was efficiently removed to form 5ʹ-hydroxyl-dsDNA, which could no longer be degraded by λ exo. Then, the dye-labeled single-stranded DNA hybridized with the 5ʹ-hydroxyl-dsDNA to form a triplex DNA (tsDNA), resulting in increased fluorescence due to the weak binding of tsDNA with the δ-FeOOH nanosheet. More importantly, a high signal-to-background ratio was obtained through magnetic separation. The detection limit for ALP was 0.02 mU/mL (3σ/S), which is much lower than that previously reported for fluorescence methods. This work revealed the application of δ-FeOOH nanosheets in the design of magnetically controlled biosensors for biomolecular detection.