(An) ultrasensitive alkaline phosphatase assay based on target-activated self-primed exponential amplification reaction

Abstract

Herein, we describe an alkaline phosphatase (ALP) assay based on Target-Activated Self-Primed Exponential Amplification (TASPA) reaction. This strategy employs a single self-primed EXPAR template (SET) containing the EXPAR template sequence extended by the self-priming sequence whose 3’ end is modified with phosphate. In the presence of the ALP activity, the SET is dephosphorylated and subjected to polymerase extension, producing a folded double-stranded DNA product (FP) with a nicking endonuclease recognition sequence. By the combined activities of DNA polymerase and nicking endonuclease, lots of trigger strands are produced, and they subsequently hybridize to the free SET, initiating further amplification reaction. As a consequence, a large number of FPs are produced as final products, which can be monitored by the fluorescence from the double strand-specific dye. Based on this strategy, we could successfully detect ALP activity down to 0.010 U/L with high selectivity toward ALP over other enzymes. The practical applicability of the developed strategy was also verified by reliably detecting the ALP activity in complex biological serum samples and the efficiency of ALP inhibitors.