Raman and fluorescence spectroelectrochemical monitoring of resazurin-resorufin fluorogenic system

Abstract Raman and fluorescence spectroelectrochemistry techniques have been used for the in-situ study of the reaction mechanism of resazurin/resorufin/dihydroresorufin system. Resazurin (RZ) is one of the most highly referenced substances used in studies on cell viability and cytotoxicity for a wide range of biological and environmental systems. Electrochemical behaviour of this tracer is based on the irreversible reduction of RZ, a slightly fluorescent compound, to resorufin (RS), a high fluorescent compound, while a subsequent and reversible reduction of RS generates dihydroresorufin (DHRS), a non-fluorescent compound. Time-resolved Raman spectroelectrochemistry has been used, for the first time, to perform dynamic studies of this system. The continuous optical acquisition times provides a large number of spectra during the whole electrochemical reaction and not only at discrete potentials, offering a novel contribution to the literature related to this tracer. The characteristic Raman and fluorescence bands associated with each molecule as well as their evolution with potential allow us to obtain valuable information about the interconversion between RZ, RS and DHRS. In addition, spectroelectrochemistry demonstrates that experimental parameters play an essential role in the fluorescent response of this coloured system. In that way, operando multiresponse techniques shed more light for the complete understanding of RZ, RS and DHRS system and they yield novel information to explain the electrochemical conversion of these compounds.