New fluorimetric NBD-derived probes for easy sensing protein carbonyls in biological medium under oxidative stress

Protein carbonylation is a key biomarker of oxidative stress involved in numerous diseases, making its accurate detection essential for understanding pathological mechanisms, developing diagnostic and therapeutic tools. The conventional 2,4-dinitrophenylhydrazine spectrophotometric probe and the more recent fluorimetric 4-hydrazino-7-nitrobenzofurazan (NBD-H) assay presents several drawbacks, including multiple washing steps. To overcome these limitations a series of N -substituted NBD probes was developed by modulating the steric and electronic properties of the hydrazone linkage. Here, we describe the synthesis and evaluation of three novel NBD-derived probes bearing methyl, ethyl, benzyl and pentyl substituents on the hydrazine moiety. Three of them formed stable hydrazone adducts with oxidized bovine serum albumin in human plasma taken as models of oxidative stress induced protein carbonyls. The data revealed that the new NBD-probes (i) exhibited stable fluorescence over time (six-fold improvement in stability), (ii) do not react with free low-molecular-weight carbonyl species, (iii) allowed to directly quantify protein carbonyls in human plasma without prior protein precipitation, (iv) offered lower limits of detection (<0.2 nmol/mg protein) under competitive conditions. Taken together, the new probes could provide a robust and simplified approach for protein carbonyl analysis in complex biological media, compatible with fluorescence microplate readers.