Spatially Quantitative Imaging of Enzyme Activity in a Living Cell

Enzyme activity plays a key role in cell heterogeneity. Its spatially quantitative imaging in a living cell not only directly displays but also helps people to understand cell heterogeneity. Current methods are hard to achieve due to the short intracellular retention or lack of internal reference of the imaging probes. Herein, we rationally designed a self-referenced Raman probe Val-Cit-Cys(StBu)-Pra-Gly-CBT (Yne-CBT) which takes an intracellular cathepsin B (CTSB)-initiated CBT-Cys click reaction to yield a long-retained cyclic dimer in cell. In the meantime, Raman signal changes of its two chemical bonds (C≡C and C≡N) after the reaction are used for self-referencing and quantitative Raman imaging of CTSB activity. In vitro experiments demonstrated that, with shell-isolated nanoparticle-enhanced Raman spectroscopy technique, 20 μM Yne-CBT was able to quantitatively detect CTSB activity with a limit of detection of 61.4 U L–1. Under a homemade microfluidic channel, Yne-CBT was successfully applied for spatially quantitative imaging CTSB activity in a living cell. Our strategy provides people with a facile method to directly and quantitatively display cell heterogeneity.