DOI:10.1021/ac400035k

Publication Date (Web): February 14, 2013

Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.