A path-choice-based biosensor to detect the activity of the alkaline phosphatase as the switch

Alkaline phosphatase (ALP), which converts the phosphate group (-PO₄) in the substrate to the hydroxyl group (-OH), is a useful tool in the biological analysis, a good indicator of dissolved inorganic phosphorus levels and an important biomarker for several diseases. In conventional designs for ALP detection, both the interferent with a -PO₄ and the target with a -OH will go into the sensing path and give out the undesired background and the desired signal respectively. This limited the sensitivity of the method and required the complicated design to achieve a satisfying limit of detection (LOD) of ALP. Here, we provided a new sensing strategy for ALP detection design. We designed a path-choice-based biosensor with two DNA tracks in which ALP works as the switch to guide the reaction path of lambda exonuclease (λ exo). The path-choice character enlarged the difference between signal and background by separating the interferent removing path and the target sensing path. The substrate preference of ALP and λ exo was studied to optimize the structure of DNA tracks. The path-choice-based biosensor achieved simple, fast (30 min), sensitive (LOD 0.014 U L^-1) and selective detection of the activity of ALP. The method has been applied to detect the activity of ALP in cell lysates, which shows the potential application in ALP-related biological research.