The assessment of unregulated level of enzyme activity is a crucial parameter for early diagnoses in a wide range of pathologies. In this study, we propose the use of electron paramagnetic resonance (EPR) as an easy method to probe carboxylesterase (CE) enzymatic activity in vitro. For this application, were synthesized two amphiphilic, nitroxide containing esters, namely Tempo‐C12 (T‐C12) and Tempo‐2‐C12 (T‐2‐C12). They exhibit low solubility in water and form stable micelles in which the radicals are EPR almost silent, but the hydrolysis of the ester bond yields narrows and intense EPR signals. The intensity of the EPR signals is proportional to the enzymatic activity. CEs1, CEs2 and esterase from porcine liver (PLE) were investigated. The obtained results show that T‐C12 and T‐2‐C12‐containing systems display a much higher selectivity toward the CEs2, with a Limit of Detection of the same order of those ones obtained with optical methods.

New stable organic radicals probes have been proposed in this study to perform Molecular Imaging by monitoring by Overhauser Magnetic Resonance Imaging (OMRI) the enzymatic activity. The radicals used are Tempo-containing esters forming stable micelles that are practically EPR silent. The hydrolysis of the ester bond catalyzed by Carboxyl esterases generates a narrow and intense EPR signal as a consequence of the release of the nitroxide radical from the micelle. Thus we have prepared a off/on probe responsive to the carboxylesterase activity that can be detected quantitatively in the OMR image.