Single-Molecule Biosensing of Alkaline Phosphatase in Cells and Serum Based on Dephosphorylation-Triggered Catalytic Assembly and Disassembly of the Fluorescent DNA Chain

Alkaline phosphatase (ALP) is a valuable biomarker and effective therapeutic target for the diagnosis and treatment of diverse human diseases, including bone disorder, cardiovascular disease, and cancers. The reported ALP assays often suffer from laborious procedures, costly reagents, inadequate sensitivity, and large sample consumption. Herein, we report a new single-molecule fluorescent biosensor for the simple and ultrasensitive detection of ALP. In this assay, the ALP-catalyzed dephosphorylation of detection probe can protect the detection probe against lambda exonuclease-mediated digestion, and the remaining detection probes can trigger ceaseless hybridization between two Cy5-labeled hairpin probes through toehold-mediated DNA strand displacement, generating a long fluorescent DNA chain, which can be subsequently separated from unhybridized hairpin probes and disassembled into dispersed Cy5 moieties upon NaOH treatment. The free Cy5 moieties indicate the presence of ALP and can be directly quantified via single-molecule counting. This biosensor enables efficient amplification and transduction of the target ALP signal through enzyme-free assembly and disassembly processes, significantly simplifying the experimental procedure and improving the assay accuracy. The proposed biosensor allows specific and ultrasensitive detection of ALP activity with a detection limit down to 2.61 × 10^–6 U mL^–1 and is suitable for ALP inhibition assay and kinetic analysis. Moreover, this biosensor can be applied for endogenous ALP detection in human cells and clinical human serum, holding the potential in the ALP biological function study and clinical diagnosis.