Butanol accelerated entropy-driven DNA walking machine for rapid and ultrasensitive determination of alkaline phosphatase activity.

Alkaline phosphatase (ALP) as an important biomarker as well as an index for the pasteurization degree of dairy food. However, there is a dilemma between the sensitivity and time-cost of ALP determination based on nucleic acid amplification approach. Herein, an ultrasensitive and rapid detection method for the ALP assay was developed based on entropy-driven DNA machine. In our design, the ALP catalyzed dephosphorylation of detection probe, which inhibited the digestion effect of lambda exonuclease. The remaining probe as a linker to tether the walking strand proximity to the surface of track strand modified gold nanoparticle, activating entropy-driven DNA machine. Accompany with walking strand moving, a large amount of assembled dye-labelled strand dissociated from gold nanoparticle with fluorescence recovery. More importantly, to further improve the walking efficiency, butanol was introduced to accelerated the signal amplification at interface, which short the incubation time from several hours to 5 min. Under the optimum condition, the change of fluorescence intensity was proportion to the concentration of ALP in the range from 0.05 U L to 5 U L with an ultralow limit of detection of 2.07 × 10 U L was achieved, which is superior to other reported methods. Furthermore, the proposed method also successfully applied for the spiked milk sample assay with satisfactory recovery in the range of 98.83%-103.00%. This work proposed a new strategy for the application of entropy-driven DNA machine in the field of rapid and ultrasensitive detection.