Ratiometric Quantification of Dissolved Molecular Oxygen in Microplates for Biochemical Assays Using Palladium...

Many biochemical processes involve the consumption and/or release of molecular oxygen (O₂). O₂-induced photoluminescence quenching of palladium-tetrapyrrol derivatives can be used to continuously measure O₂ concentrations in biochemical reactions and during microbial growth in standard microtiter plates (MTPs). Palladium-(II)-5,10,15,20-(tetrapentafluorophenyl)-porphyrin (1; CAS 72076-09-6) and Palladium-(II)-5,10,15,20-(tetraphenyl)-tetrabenzoporphyrin (2; CAS 119654-64-7) are introduced with this study. Spectral analyses of both compounds revealed that photoluminescence quenching by O₂ is not evenly distributed throughout all wavelengths and can therefore be used ratiometrically. Experimentally determined photoluminescence lifetimes are around 500 and 300 μs for 1 and 2, respectively. A simple protocol is disclosed on how to immobilize the indicators on the bottom of MTP wells to give transparent, dye-doped polymer layers. We propose a straightforward procedure for how photoluminescence data can be processed and calibrated in terms of O₂ concentrations. Diverse applications are demonstrated and discussed, which include oxygen consumption and production by microorganisms as well as by enzymatically catalyzed biochemical reactions. Various aspects are critically considered, such as the dependence of O₂ solubility on temperature and salinity, the diffusion of O₂ across diverse phase boundaries, the unwanted O₂ ingress into the reaction mixture, the oxygen binding capacity of the MTP plastic material, and the pH dependence of the sensor layer. The findings and methods presented here open up a wide variety of high throughput assays involving changes of dissolved O₂ as measurands for biochemical and biological activity.