A specific esterase and pH logically regulate ESIPT: different kinds of granulocyte sorting

Area of Science:

• Analytical Chemistry
• Biomedical Engineering
• Fluorescence Spectroscopy

Background:

• Accurate granulocyte differentiation is crucial for blood analysis and clinical diagnostics.
• Simultaneous detection of naphthol AS-D chloroacetate esterase (NAS-DCE) and pH offers a promising approach for granulocyte separation.

Purpose of the Study:

• To design and synthesize novel fluorescent small molecules (HBT-ASDs) capable of responding to both NAS-DCE activity and pH.
• To establish a method for real-time monitoring and differentiation of granulocytes based on their NAS-DCE activity and intracellular pH.

Main Methods:

• Design and synthesis of a series of fluorescent small molecules (HBT-ASDs) exhibiting Excited-State Intramolecular Proton Transfer (ESIPT).
• Utilized HBT-ASD-2, a representative molecule, to detect NAS-DCE activity across different pH values (5.0, 7.4, 10).
• Analyzed fluorescence emission spectra at 438 nm and 545 nm to correlate with specific granulocyte types and NAS-DCE activity.

Main Results:

• HBT-ASD-2 demonstrated distinct fluorescence outputs at 438 nm and 545 nm, varying with NAS-DCE activity and pH.
• The developed fluorescent probe enabled the differentiation and sorting of acid, neutrophil, and alkaline granulocytes.
• Real-time monitoring of NAS-DCE activity was achieved, providing quantitative insights into enzyme function.

Conclusions:

• The designed HBT-ASDs offer a versatile platform for simultaneous detection of NAS-DCE and pH.
• This approach provides a simple yet effective analytical tool for clinical blood monitoring and granulocyte analysis.
• The study highlights the potential of tailored fluorescent probes in advancing diagnostic capabilities for hematological disorders.