Determine precise Oxygen concentration in synchrony with optical spectroscopy measurements


Measurement of oxygen (O2) is an important assay in biochemistry to monitor oxidation/reduction reactions, oxidative phosphorylation, myoglobin / hemoglobin reactions, and photosynthesis to name a few. The challenge of this measurement is to be able to reliably detect small changes in O2 at low concentrationin a suspension of cells. A fast response is critical to obtain enough data points to characterize the reaction and to identify these small changes within the timespan of the experiment.  Suspended cells complicate optical measurement due to scattering. This lead to the use of time–resolved fluorophores


Monitor Oxidation/Reduction reactions
Oxidative Phosphorylation
Myoglobin/Hemoglobin reactions

Oxygen Electrodes are subject to changes in diffusion resistance

Electrode probes also suffer from electrical interference, and since the consume oxygen, can easily generate misleading data. This also requires extensive re-calibration to remove residual current generated by the electrochemical reaction, when when no oxygen is present.

Electrode limitations include a slower response, more drift, and require maintenance, in addition to requiring specialized serialization procedures and periodic reconditioning.

Yutaka Amao, Probes and Polymers for Optical Sensing of Oxygen, Microchimica Acta, September 143 (2003) 1-12.

A smarter solution

We use Phosphorescence Quenching, in which the phosphor is placed is placed in the medium of interest and then excited by absorbing a photon of light emitted from an LED. After absorption, the phosphor then returns to it’s ground state by emitting the light that was just absorbed,or by a non-radiative transfer of energy to other “quenching” molecules in the environment.

The quenching of phosphorescence by oxygen is determined by the frequency of excited triplet state molecules and oxygen. Phosphorescence is much more sensitive to oxygen measurement than fluorescence because it has much longer lifetime and therefore has a higher quenching probability than long-lived fluorescence. The longer lifetime allows one to more easily use the pulse method to measure oxygen accurately by increasing the number of measurements to fit the Stern-Volmer relationship.

OLIS uses a precise technique for oxygen measurement which employs an oxygen-dependent quenching of phosphorescence using a new generation of dyes by Oxygen Enterprises

The OLIS process for oxygen measurement determines the oxygen concentration directly by determining the phosphorescence lifetime instead of intensity. Intensity is a relative measurement while the lifetime is an intrinsic molecular property which is (for the most part) independent of concentration.

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