A ratiometric fluorescent assay for the detection and bioimaging of alkaline phosphatase based on near infrared Ag₂S quantum dots and calcein

Herein, a ratiometric fluorescent method was developed for alkaline phosphatase (ALP)detection based on near-infrared (NIR)Ag₂S quantum dots (QDs)and calcein through the competitive approach. The system based on Ag₂S QDs and calcein shows green (maximum emission at 512 nm from calcein)and near infrared (NIR)fluorescence (maximum 798 nm from Ag₂S QDs)under the same excitation wavelength (468 nm). In the presence of Ce³⁺, the fluorescence intensity of calcein is decreased due to static quenching, while the fluorescence intensity of Ag₂S QDs is enhanced through aggregation induced emission (AIE). The p-nitrophenyl phosphate is hydrolyzed by ALP, and the yield phosphate ions bind with Ce³⁺ with higher affinity than these of Ag₂S QDs and calcein. Therefore, the green fluorescence from calcein is recovered while NIR fluorescence from Ag₂S QDs is decreased. On the basis of these findings, a ratiometric fluorescence assay was developed for the measurement of ALP activity. The ratio of fluorescence intensity at 512 and 798 nm (F₅₁₂/F₇₉₈)was well associated with the ALP concentration ranging from 2 to 100 mU/mL with the detection limit of 1.28 mU/mL. The method was successfully applied for detecting ALP in human serum with an acceptable recovery and bioimaging intracellular ALP with good performance. In addition, the approach was also employed for the screening ALP inhibitor.