Ultrahigh Efficient FRET Ratiometric Fluorescence Biosensor for Visual Detection of Alkaline Phosphatase Activity and Its Inhibitor

Herein, a ratiometric fluorescence sensor based on the fluorescence resonance energy transfer (FRET) strategy was proposed to detect alkaline phosphatase (ALP) activity and its inhibitor (sodium vanadate, Na3VO4). The principle was that blue fluorescent nitrogen and sulfur co-doped carbon dots (N/S-CDs) were designed as energy donors, and ALP induced the formation of yellow fluorescent resultant 2,3-diaminophenazine (DPA) as an energy acceptor, causing the fluorescence to change from blue to yellow. Indeed, the FRET efficiency reached up to 93.2%, which is higher than most reported FRET strategies for sensing and imaging. After the recognition of ALP, the fluorescence intensity achieved a ratiometric change, accompanied by a significant transition in fluorescence color (Stokes wavelength shifted about 220 nm), which can achieve quantitative detection with a limit of detection being 0.396 U/L and visual qualitative analysis of ALP activity. Moreover, dual-emission self-correction ability can reduce external interference and improve the stability. The strong coordination ability between pyrophosphate (PPi) and Cu2+ and the efficient hydrolysis of PPi by ALP determined the specificity of the biosensor, and satisfactory results were obtained in the detection of clinical human serum samples. The proposed ratiometric fluorescence biosensor provides an alternative way for effective evaluation of ALP-related diseases, and it can also be used for the assessment of Cu2+, PPi, and Na3VO4 content and the screening of ALP inhibitors.