Spatially Quantitative\nImaging of Enzyme Activity\nin a Living Cell

Enzyme activity plays a key role\nin cell heterogeneity.\nIts spatially\nquantitative imaging in a living cell not only directly displays but\nalso helps people to understand cell heterogeneity. Current methods\nare hard to achieve due to the short intracellular retention or lack\nof internal reference of the imaging probes. Herein, we rationally\ndesigned a self-referenced Raman probe Val-Cit-Cys(StBu)-Pra-Gly-CBT\n(<b>Yne-CBT</b>) which takes an intracellular cathepsin B (CTSB)-initiated\nCBT-Cys click reaction to yield a long-retained cyclic dimer in cell.\nIn the meantime, Raman signal changes of its two chemical bonds (CC\nand CN) after the reaction are used for self-referencing and\nquantitative Raman imaging of CTSB activity. In vitro experiments\ndemonstrated that, with shell-isolated nanoparticle-enhanced Raman\nspectroscopy technique, 20 μM <b>Yne-CBT</b> was able\nto quantitatively detect CTSB activity with a limit of detection of\n61.4 U L<sup>–1</sup>. Under a homemade microfluidic channel, <b>Yne-CBT</b> was successfully applied for spatially quantitative\nimaging CTSB activity in a living cell. Our strategy provides people\nwith a facile method to directly and quantitatively display cell heterogeneity.