P.Lefrançois, V. S. R. Vajrala, I. B. Arredondo, B. Goudeau,Th. Doneux, L. Bouffier,
S. Arbault

Phys. Chem. Chem. Phys., 2016, 18, 25817-25822.

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 (labs =
572 nm) and yields strong fluorescence (lfluo = 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.

Graphical Abstract