Multicolor Activatable Raman Probes for Simultaneous Detection of Plural Enzyme Activities

Abstract

Raman probes based on alkyne or nitrile tags hold promise for highly multiplexed imaging. However, sensing of enzyme activities with Raman probes is difficult because few mechanisms are available to modulate the vibrational response. Here we present a general strategy to prepare activatable Raman probes that show enhanced Raman signals due to electronic preresonance (EPR) upon reaction with enzymes under physiological conditions. We identified a xanthene derivative bearing a nitrile group at position 9 (9CN-JCP) as a suitable scaffold dye, and synthesized four types of activatable Raman probes, which are targeted to different enzymes (three aminopeptidases and a glycosidase) and tuned to different vibrational frequencies by isotope editing of the nitrile group. We validated the activation of the Raman signals of these probes by the target enzymes and succeeded in simultaneous imaging of the four enzyme activities in live cells. Different cell lines showed different patterns of these enzyme activities.

9-Cyanopyronin probe palette for super-multiplexed vibrational imaging

“…Indeed, a large number of interesting sensors have been developed based on xanthenes 29 , 38 , 39 , the parent structures of MARS probes. In fact, a very recent work reported MARS-derivatized probes for detecting enzyme activities 40 . Hence, we expect MARS probes to make important contribution to the multiplexed sensing applications.…”

A decade of alkyne-tag Raman imaging (ATRI): applications in biological systems

“…By using isotopically edited nitrile they were able to simultaneously detect four different enzymes. 34 Inherent nitrile in molecules like anticancer drug neratinib, plant fungicides azoxystrobin and chlorothalonil have been used for imaging their distribution. 77,78 Most recently, photoswitchable Raman probes with nitrile groups have been reported that can be turned on and off with different wavelengths of light.…”

“…33 This technique has huge potential in biological imaging. 34 Advances in SRS imaging techniques and applications have been reviewed elsewhere. [35][36][37][38][39] 1.1.3 Coherent anti-Stokes Raman scattering.…”

Toward Raman Subcellular Imaging of Endothelial Dysfunction

“…Copyright 2013 Royal Society of Chemistry. Panel (C) is adapted from ref ( 89 ). Copyright 2020 American Chemical Society.…”

“…Overall, the detected enzyme activities were in agreement with their expression levels as assessed by PCR. 89 …”