title: Direct oxidative pathway from amplex red to resorufin revealed by in situ confocal imaging.
abstract: Amplex Red (AR) is a very useful chemical probe that is employed in biochemical assays. In these assays, the non-fluorescent AR is converted to resorufin (RS), which strongly absorbs in the visible region (λabs = 572 nm) and yields strong fluorescence (λfluo = 583 nm). Even if AR is commonly used to report on enzymatic oxidase activities, an increasing number of possible interferences have been reported, thus lowering the accuracy of the so-called AR assay. As a redox-based reaction, we propose here to directly promote the conversion of AR to RS by means of electrochemistry. The process was first assessed by classic electrochemical and spectroelectrochemical investigations. In addition, we imaged the electrochemical conversion of AR to RS at the electrode surface by in situ confocal microscopy. The coupling of methodologies allowed to demonstrate that RS is directly formed from AR by an oxidation step, unlike what was previously reported. This gives a new insight in the deciphering of AR assays' mechanism and about their observed discrepancy.
authors.affiliation: Univ. Bordeaux, ISM, UMR 5255, F-33400 Talence, France. laurent.bouffier@enscbp.fr stephane.arbault@enscbp.fr.
authors.forename: Pauline
authors.initials: P
authors.lastname: Lefrançois
authors.forename: Venkata Suresh Reddy
authors.initials: VS
authors.lastname: Vajrala
authors.forename: Imelda Bonifas
authors.initials: IB
authors.lastname: Arredondo
authors.forename: Bertrand
authors.initials: B
authors.lastname: Goudeau
authors.forename: Thomas
authors.initials: T
authors.lastname: Doneux
authors.forename: Laurent
authors.initials: L
authors.lastname: Bouffier
authors.forename: Stéphane
authors.initials: S
authors.lastname: Arbault