Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Imaging

Background:

• Simultaneous detection of multiple enzyme activities is crucial for understanding complex biological processes.
• Raman imaging offers high multiplexing capabilities for biological analysis.
• Previous Raman probes based on 9CN-pyronins faced challenges with hydrolysis product leakage.

Purpose of the Study:

• To develop novel Raman probes for enzyme activity detection with improved spatial specificity and signal retention.
• To establish a new Raman signal activation mechanism combining aggregate formation and resonant Raman effects.
• To enable simultaneous visualization of multiple enzyme activities in live cells and ex vivo tissues.

Main Methods:

• Development of 9CN-rhodol derivatives, specifically 9CN-JCR, for Raman probe applications.
• Utilizing aggregate formation and resonant Raman effects for signal activation and enhancement.
• Employing isotope-edited 9CN-JCR-based probes for simultaneous multi-enzyme activity detection.

Main Results:

• The 9CN-JCR derivative demonstrated enhanced stimulated Raman scattering (SRS) signal intensity and aggregate-forming ability.
• Probes exhibited improved retention of hydrolysis products within target cells after activation.
• Simultaneous detection of β-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities was achieved in live cells.
• Enzyme activity regions were distinguished in Drosophila wing disc and fat body ex vivo.

Conclusions:

• Novel 9CN-rhodol-based Raman probes enable robust, simultaneous detection of multiple enzyme activities.
• The developed probes offer enhanced signal intensity and retention for precise biological imaging.
• This approach advances the capability of Raman imaging for investigating cellular enzyme functions.