Dual-factor Synergistically Activated ESIPT-based Probe: Differential Fluorescence Signals to Simultaneously Detect α-Naphthyl Acetate and Acid α-Naphthyl Acetate Esterase

alpha-Naphthyl acetate esterase (alpha-NAE) and acid alpha-naphthyl acetate esterase (ANAE), a class of special esterases, are important for lymphocyte typing and immunocompetence-monitoring. As such, the simultaneous detection of alpha-NAE and ANAE has become a target to effectively improve the accuracy in lymphocyte typing. Therefore, we developed a dual-factor synergistically activated ESIPT-based probe (HBT-NA) to detect alpha-NAE and ANAE sensitively, rapidly, and simultaneously in a differential manner. HBT-NA exhibits differential fluorescence signal outputs toward small changes of alpha-NAE and ANAE activities. HBT-NA displays a weak fluorescence signal at 392 nm over a pH range from 6.0 to 7.4. However, when it interacts with alpha-NAE (0-25 U) at pH = 7.4, the fluorescence intensity at 392 nm enhanced linearly within 60 s (F-392 nm/F0(392 nm) = 0.042 C alpha-NAE + 1.1, R-2 = 0.99). Furthermore, HBT-NA emits ratiometric fluorescence signals (F-505 nm/F-392 (nm)) for ANAE (0-25 U) at pH = 6.0 within 2.0 min, exhibiting a good linear relationship (F-505 nm/F-392 (nm) = 0.83C(ANAE) - 1.75, R-2 = 0.99). The differential fluorescence signals can be used to simultaneously detect the activities of alpha-NAE and ANAE in solutions and complex living organisms. More importantly, based on the differential fluorescence signals toward alpha-NAE and ANAE, T lymphocytes and B lymphocytes could be successfully typed and differentiated among nontyped lymphocytes, facilitating the real-time evaluation of their immune functions using flow cytometry. Hence, HBT-NA could be used for the ultrasensitive detection of the enzyme activities of alpha-NAE and ANAE, the real-time precise typing of lymphocytes, and the monitoring of immunocompetence.

Automated summary

The newly developed HBT-NA probe enables sensitive, rapid, and differential detection of alpha-NAE and ANAE activities, providing a new approach to improve the accuracy of lymphocyte typing.