Exploring the tunable excitation of QDs to maximize the overlap with the absorber for inner filter effect-based phosphorescence sensing of alkaline phosphatase.

The inner filter effect (IFE) is an effective way for fluorescence modulation and thus has been extensively explored for the development of fluorescence assays. Theoretically, the key to maximize the sensitivity of IFE-based fluorescence assays is to enlarge the overlap between the absorption of the absorber and the excitation/emission of the fluorophore. Therefore, in this work, the tunable excitation of quantum dots (QDs) was explored for screening of the IFE pair having the best IFE-based assay sensitivity. A series of QDs, including CdTe QDs with different sizes, carbon dots, Cu-doped CdS QDs, and Mn-doped ZnS QDs, were investigated. PNPP (p-nitrophenylphosphate) was chosen as the absorber since its absorption overlapped with the above QDs. Besides, it can be catalytically converted to p-nitrophenol (PNP) by alkaline phosphatase (ALP) together with an absorption spectrum change (red-shift). Interestingly, it was found that the IFE efficiency of different PNPP-QD pairs increased almost linearly with the corresponding spectral overlap, and Mn-doped ZnS QDs were eventually chosen for the IFE assay of ALP because of the maximum spectral overlap and thus the best sensitivity. A simple and sensitive turn-on phosphorescence ALP assay was developed, with a detection limit of 4 × 10-4 U L-1. Because of the high sensitivity, we also found that ALP of different origins possessed different enzymatic activities. The developed ALP phosphorescence assay was successfully employed for the analysis of ALP in serum samples.