A colourimetric sensor for the simultaneous determination of oxidative status and antioxidant activity on the same membrane: N,N-dimethyl-p-phenylene diamine (DMPD) on Nafion.

A colourimetric sensor capable of simultaneously measuring oxidative status (OS) in terms of the hazard produced by reactive oxygen species (ROS) and antioxidant activity (AOA) in regard to ROS-scavenging ability of antioxidant compounds was developed. The coloured cationic semi-quinone derivatives, caused by ROS oxidative degradation of N,N-dimethyl-p-phenylene diamine hydrochloride (DMPD) in pH 5.7 acetate-buffered medium, were formed in solution and immobilized on a perfluorosulfonate-based Nafion membrane. ROS, namely hydroxyl (·OH) and superoxide (O2(·-)) radicals, were produced by Fenton/UV and xanthine/xanthine oxidase methods, respectively. The pink-coloured, (+)-charged chromophore (referred to as DMPD-quinone or DMPDQ), resulting from the reaction between DMPD and ROS, could be completely retained on the solid membrane sensor by electrostatic interaction with the anionic sulfonate groups of Nafion. After equilibration, the Nafion membrane surface was homogeneously coloured enabling an absorbance measurement at 514 nm, while the aqueous phase completely lacked colour. Antioxidants, when present, caused an absorbance decrease on the membrane due to their ROS scavenging action, giving rise to less DMPDQ production. The absorbance decrease on the sensor was linearly dependent on antioxidant concentration over a reasonable concentration range, enabling the simultaneous determination of OS and AOA-against ROS. The proposed antioxidant sensing method was tested in synthetic and real antioxidant mixtures, and validated against standard antioxidant capacity assays (i.e. ABTS and CUPRAC) for a variety of polyphenolic and antioxidant compounds. The dynamic linear ranges of antioxidants with the DMPD sensor in protection against hydroxyl and superoxide radicals generally varied within the micromolar to a few tens of micromolar concentration interval over one order-of-magnitude. Choosing three representative compounds in the high (epigallocatechin gallate), medium (quercetin) and low (p-coumaric acid) molar absorptivity range, the detection limits ranged within the concentration intervals of 0.2-0.9 μM, 0.3-0.8 μM, and 4-14 μM, respectively, depending on the radical scavenged.