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 (l abs = 572 nm) and yields strong fluorescence (l fluo = 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.

A snapshot of the electrochemical reaction layer by using 3 dimensionally resolved fluorescence mapping

“…For example, it allowed the determination of the number of electrons involved in the electrochemical reduction of O 2 , 16 or deciphering the oxidative mechanism responsible for Amplex® Red fluorescence by in situ formation of RF. 17 In this work, we demonstrate that EC-FCLSM is well-suited to probe the chemical reactivity inside the diffusion layer. For that, we selected again RZ and RF dyes which are very important molecular probes used in many bioassays ( Scheme 1 ).…”

Importance of radiolytic reactions during high-LET irradiation modalities: LET effect, role of O2 and radiosensitization by nanoparticles

“…The fluorescent probe we chose, Ampliflu ® Red, allowed an extremely sensitive (Baldacchino et al 2009;Foley et al 2005;Maeyama et al 2011b) in situ and in operando detection without any separation step of molecular products, via chromatography for example. This molecule is known to react with hydrogen peroxide in combination with HRP enzyme and is commonly used to detect oxidant species in microbial systems (Castaño et al 2018) or under stress conditions (Lefrancois et al 2016;Kovacik and Babula 2017). This dye and derivatives were also used as chemical dosimeter in proton irradiation (Kim et al 2007).…”

Wireless Addressing of Freestanding MoSe2 Macro- and Microparticles by Bipolar Electrochemistry

“…Here, the well-known Amplex® Red molecule (AR) was selected to exemplify this concept. [33][34][35] AR is a commercially available chromo-and fluorogenic probe that is widely used in many bioassays. The molecule is colorless in its initial form, but a deep pink coloration (λabs = 570 nm, ε = 51,000 M -1 cm -1 ) and high fluorescence (λfluo = 592 nm, ϕ = 0.41) is observed upon oxidation to resorufin dye (RF).…”

“…28 Briefly, two feeder electrodes were placed in plastic vials that were connected together by a glass capillary (Ø = 6 mm). The distance between the feeder electrodes was 35 Author's version of the Accepted Manuscript published in Journal of Physical Chemistry C 123 (2019) 5647-5652, doi: 10.1021/acs.jpcc.8b09702. https://pubs.acs.org/doi/10.1021/acs.jpcc.8b09702…”