Dual-factor Synergistically Activated ESIPT-based Probe: Differential Fluorescence Signals to Simultaneously Detect...

α-Naphthyl acetate esterase (α-NAE) and acid α-naphthyl acetate esterase (ANAE), a class of special esterases, are important for lymphocyte typing and immunocompetence-monitoring. As such, the simultaneous detection of α-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 α-NAE and ANAE sensitively, rapidly, and simultaneously in a differential manner. HBT-NA exhibits differential fluorescence signal outputs toward small changes of α-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 α-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 + 1.1, R² = 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 - 1.75, R² = 0.99). The differential fluorescence signals can be used to simultaneously detect the activities of α-NAE and ANAE in solutions and complex living organisms. More importantly, based on the differential fluorescence signals toward α-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 α-NAE and ANAE, the real-time precise typing of lymphocytes, and the monitoring of immunocompetence.