A reversible polyphenol oxidase-ABTS colourimetric response mediated by Scedosporium apiospermum for visual detection of oxygen ingress under anaerobic conditions.

Rapid and reliable verification of strict anaerobic conditions is essential for the cultivation, purification, and physiological studies of anaerobes, as well as for several chemical, material, and biotechnological workflows where oxygen ingress can significantly disrupt process integrity. Existing oxygen indicators are limited by a lack of specificity, reversibility, spatial relevance, and temporal resolution, underscoring the need to identify and develop highly sensitive, accurate, reliable, low-maintenance, and cost-effective oxygen-sensing methods. Here, we examined whether the intrinsic oxygen-dependent oxidation of the chromogenic substrate ABTS by the polyphenol oxidase (PPO) class of enzymes, together with the redox reversibility of its bluish-green oxidized metastable product, could be repurposed as a simple, qualitative and visually interpretable indicator of oxygen ingress into anaerobic systems. Using the facultatively anaerobic fungus Scedosporium apiospermum, ABTS oxidation was examined under controlled aerobic and anaerobic conditions. The results showed that ABTS oxidation was oxygen-dependent; removal of oxygen resulted in a gradual loss of coloration, consistent with cessation or reduction of oxidative regeneration and subsequent decay of the preformed oxidized product. Transitions between aerobic and anaerobic conditions produced corresponding reversible changes in color intensity, potentially enabling both real-time detection and time-integrated recording of oxygen exposure. These findings provide proof-of-concept evidence that the classical PPO-ABTS enzymatic assay could be repurposed as a simple, visually interpretable, reversible indicator of anaerobic integrity, offering a low-cost and instrumentation-free complement to existing anaerobic monitoring approaches in microbiological and biotechnological applications.