Fluorescent Light-up Probe with Aggregation-Induced Emission Characteristics for Alkaline Phosphatase Sensing and Activity Study

Fluorogens with aggregation-induced emission (AIE) characteristics have attracted intensified research interest in biosensing applications, and those with specific targeting ability are especially desirable. In this work, we designed and synthesized an AIE fluorescent probe by functionalizing a tetraphenylethylene (TPE) fluorogen with two phosphate groups (TPE-phos) for the detection of alkaline phosphatase (ALP) and its enzymatic activity based on the specific interaction between the probe and ALP. The probe is virtually nonfluorescent in aqueous media due to good water solubility. In the presence of ALP, the phosphate groups are cleaved through enzymatic hydrolysis, yielding a highly fluorescent product as a result of activated AIE process. This light-up probe shows excellent selectivity toward ALP among a group of proteins. The detection limit is found to be 11.4 pM or 0.2 U L(-1) in Tris buffer solution with a linear quantification range of 3-526 U L(-1). The assay is also successfully performed in diluted serum with a linear range up to 175 U L(-1), demonstrating its potential application in clinical analysis of ALP levels in real samples. Furthermore, by conducting kinetic analysis of the enzyme using TPE-phos as the substrate, the kinetic parameter kcat/KM is determined to be 5.1×10(5) M(-1) s(-1), indicating a high efficiency of the substrate.