Ratiometric detection of alkaline phosphatase based on aggregation-induced emission enhancement

Alkaline phosphatase (ALP) is an important enzyme that is associated with many human diseases, so the quantitative detection of ALP is vital from a clinical perspective. Nevertheless, most fluorescent assays for monitoring ALP depend on aggregation-induced quenching (ACQ), single-signal modulation, or a "signal off" mode, which suffer from poor sensitivity, a "false positive" problem, and low signal output. In this work, we utilized the electrostatically driven self-assembly of glutathione-capped gold nanoclusters (GSH-AuNCs, which show aggregation-induced emission, AIE) and amino-modified silicon nanoparticles (SiNPs) to create a hybrid probe (SiNPs@GSH-AuNCs). This nanohybrid probe showed emission from the SiNPs at around 470 nm as well as aggregation-induced emission enhancement (AIEE) of the GSH-AuNCs at 580 nm. The AIEE of the GSH-AuNCs was quenched in the presence of KMnO₄, but the AIEE was recovered by adding ascorbic acid as an oxidation-reduction reaction occurred between KMnO₄ and the ascorbic acid. The fluorescence of the SiNPs remained constant whether the AIEE was quenched or not, meaning that the fluorescence of the SiNPs could be used as an internal reference. In a typical enzymatic reaction, ascorbic acid 2-phosphate is hydrolyzed by ALP to produce ascorbic acid. Therefore, the hybrid probe was shown to allow the ratiometric detection of ALP, with a linear range of 0.5-10 U L^-1 and a limit of detection (LOD) of 0.23 U L^-1. Finally, the proposed analytical strategy was successfully applied to detect ALP in human serum samples and to determine the concentration of an ALP inhibitor. Graphical Abstract.