A transformer of molecular beacon for sensitive and real-time detection of phosphatases with effective inhibition of the false positive signals

Molecular beacons (MBs) have shown great potential in measurement of enzyme activities. However, currently available methods for monitoring of phosphatases only use MBs as a signal reporter. Extra substrates for the phosphatases are needed to hybridize to the MB either as a primer or as a template. Moreover, few MB-based methods have been used to detect enzyme activities in real biological samples due to insufficient sensitivity or false positive interference signals caused by nonspecific nucleases. In this work, a novel type of fluorescent probe was designed and synthesized for monitoring of phosphatases by integrating the DNA substrate and the signaling structures into a single molecule. Such a new design not only significantly simplified the probing system and greatly enhanced the sensitivity, but also offered a practical way to guard against the false-positive signal problems in the application to real samples. The unique design of the assay format should be widely applicable to many other enzymatic assays using oligonucleotide fluorescent probes.