Highly Sensitive “Off/On” EPR Probes to Monitor Enzymatic Activity

Abstract

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.

4-Amino-TEMPO loaded liposomes as sensitive EPR and OMRI probes for the detection of phospholipase A2 activity

“…On this basis, our work aimed at developing an in vitro diagnostic EPR method based on the measurements of stable 4-amino-TEMPO (TMN) radicals released by “EPR silent” liposomes thanks to PLA2 activity. EPR enzymatic assays were already reported in the literature 28 – 30 but this is the first example that deals with a liposomal on/off EPR probe for the PLA2 activity detection. Finally, the use of radical-based probes may open new avenues for in vivo detection of the enzyme activity in a pathological tissue by means of in vivo Overhauser-enhanced Magnetic Resonance Imaging (OMRI) 31 – 36 .…”

EPR-basedin situenzymatic activity detection of endogenous caspase-3 in apoptosis cell lysates

“…In recent years, EPR has also emerged as a suitable tool for analytical studies because of its high sensitivity (up to 10 À9 -10 À10 mol for solids and 10 À13 -10 À14 mol for solutions, under optimized conditions), high specificity for paramagnetic species, and detection in a noninvasive manner, features which are superior to those of many other analytical methods. [17][18][19][20] Also, EPR spectroscopy has advantages such as the absence of endogenous background signals and simple experimental procedure, offering a highly promising route for the detection and characterization of apoptosis-related proteases under ambient conditions. 21 In this work, we report an EPR-based enzymatic assay for reliable and rapid detection of caspase-3 activity in cell apoptosis.…”