A Double-Locked ESIPT-AIE Fluorescent Probe Detects Esterase with Highly Matched Response Kinetics

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Hydrolases are crucial in biological processes; their dysfunction is linked to human diseases.
• In situ fluorescence detection offers spatiotemporal analysis of hydrolase activity but faces limitations in imaging fidelity.
• Current methods often capture only specific enzyme-probe interaction stages, leading to suboptimal results.

Purpose of the Study:

• To develop an advanced activatable fluorescent probe for accurate hydrolase activity detection.
• To address limitations of current methods by considering the entire enzymatic response kinetics.
• To create a probe with enhanced specificity and reduced background noise for reliable imaging.

Main Methods:

• Development of a double-locked Excited State Intramolecular Proton Transfer-Aggregation Induced Emission (ESIPT-AIE) fluorescent probe (Br-3N-2Et).
• Validation of the probe's mechanism through enhanced pre-reaction recognition and reduced basal fluorescence.
• Assessment of the probe's affinity, binding kinetics, and Stokes shift via interactions with hydrolase catalytic sites.
• Demonstration of dual-emission mechanism for rapid signal enhancement and in situ imaging.

Main Results:

• The Br-3N-2Et probe exhibited reduced basal fluorescence (Φ = 0.0183) and increased resistance to interference.
• Enhanced affinity for hydrolase catalytic sites and a significant Stokes shift (188 nm) were observed.
• The ESIPT-AIE mechanism enabled a 132.2-fold in situ fluorescence signal enhancement.
• Regional differential detection of esterase activity in HepG2 cells and endometrial cancer tissues was achieved.

Conclusions:

• The developed double-locked ESIPT-AIE probe accurately monitors hydrolase activity by encompassing the entire enzymatic response.
• This probe offers improved imaging fidelity and reliability compared to existing methods.
• The findings pave the way for integrated, multimechanism platforms for hydrolase sensing in complex biological systems.