Spatially Quantitative Imaging of Enzyme Activity in a Living Cell

Area of Science:

• Biochemistry
• Cell Biology
• Chemical Biology

Background:

• Enzyme activity is crucial for understanding cell heterogeneity.
• Existing imaging probes have limitations like short retention and lack of internal references.
• Spatially quantitative imaging of enzyme activity in living cells is challenging.

Purpose of the Study:

• To design a self-referenced Raman probe for quantitative imaging of intracellular cathepsin B (CTSB) activity.
• To overcome the limitations of current imaging probes for assessing cell heterogeneity.
• To enable direct and quantitative display of cell heterogeneity through enzyme activity imaging.

Main Methods:

• Rational design of a self-referenced Raman probe, Yne-CBT.
• Utilizing an intracellular cathepsin B (CTSB)-initiated click reaction for probe activation and retention.
• Employing shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) for in vitro detection.
• Applying a microfluidic channel for spatially quantitative imaging in living cells.

Main Results:

• The Yne-CBT probe undergoes a click reaction initiated by CTSB, forming a long-retained cyclic dimer.
• Raman signal changes in the probe's C≡C and C≡N bonds allow for self-referencing and quantitative imaging.
• In vitro, Yne-CBT achieved a limit of detection of 61.4 U L⁻¹ for CTSB activity.
• Successful spatially quantitative imaging of CTSB activity in living cells was demonstrated.

Conclusions:

• The developed Yne-CBT probe provides a self-referenced and quantitative method for imaging CTSB activity.
• This strategy effectively addresses the limitations of probe retention and internal referencing in cellular imaging.
• The approach offers a facile method for directly and quantitatively displaying cell heterogeneity via enzyme activity.