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

Abstract Many biochemical processes are dependent on the presence or absence of molecular oxygen (O2). Palladium-tetrapyrrol derivatives can be used to measure O2-concentrations and O2-turnover during biochemical reactions and 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 fluorescence quenching by O2 is not evenly distributed throughout all wavelengths and can therefore be used ratiometrically. Experimentally determined fluorescence lifetimes are around 500 µs and 300 µs for 1 and 2, respectively. A simple protocol is disclosed, how to immobilize the indicators on the bottom of MTP wells to give clear transparent dye doped polymer layers. We propose a straightforward procedure of how fluorescence 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 catalysed biochemical reactions. Various aspects are critically considered, as there are e.g. the dependence of O2 solubility on temperature and salinity, the diffusion of O2 across diverse phase boundaries, the unwanted O2 ingress into the reaction volume, 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 broad variety of high throughput assays involving changes of dissolved O2 as measurands for biochemical and biological activity. Competing Interest Statement The authors have declared no competing interest. Abbreviations - 1 - Palladium(II)-5,10,15,20-(tetrapentafluorophenyl)porphyrin (CAS 72076-09-6) - 2 - Palladium(II)-5,10,15,20-(tetraphenyl)tetrabenzoporphyrin (CAS 119654-64-7) - [M] - a molecular species M in brackets designates the concentration in the solution given - Avr - average - a.U. - arbitrary units - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EtOH - Ethanol - FWHM - full width at half maximum - HTP - high throuput - KPB - potassium phosphate buffer - MTP - microtiterplate - MW - molecular weight - OD - optical density - NMP - noble metal porphyrin - PAR - photosynthetic active radiation - PS - polystyrene - SD - standard deviation - SI - supporting information - S-PS - sulfonated polystyrene Variable Declaration - τ (greek letter: tau) - time constant - θ (greek letter: theta) - temperature - F - fluorescence - R - Ratio of fluorescence intensities - T - integration time - t - time