An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence.

An optical method for measuring oxygen concentrations in aqueous solutions is described. This method is based upon the oxygen-dependent quenching of phosphorescence. Phosphorescence excitation and emission spectra and lifetimes of some of the probe molecules suitable for measurement of oxygen in aqueous solutions are given. The probes include fluorescein derivatives, 4'5'-diiodofluorescein, eosin Y, 5(and 6)-carboxyeosin, erythrosin, and 5(and 6)-carboxyerythrosin as well as the Zn(II), Y(III), Sn(IV), Lu(III), and Pd(II) derivatives of meso-tetra-(4-sulfonatophenyl)-porphine, meso-tetra-(N-methyl-4-pyridyl)-porphine and coproporphyrin. The phosphorescence lifetimes of the given probes were found to depend upon the oxygen concentration by a simple Stern-Volmer relationship with a quenching constant of approximately 10(9) M-1 S-1. Binding of the molecules to bovine serum albumin decreased the quenching constant for oxygen by approximately an order of magnitude and also inhibited probe self-quenching, indicating that at the protein binding site the probes are somewhat protected from collision with quenchers. The use of this optical method for measuring oxygen is demonstrated for reactions catalyzed by glucose oxidase and by cytochrome c oxidase. It is shown that, using this method oxygen concentrations can be measured from approximately 250 microM (air saturation) down to the nanomolar range.