# how to measure dissolved oxygen?

How do you measure dissolved oxygen reliably in a culture of cells? This page has info on one sensor: http://water.me.vccs.edu/concepts/domeasure.html

how would you know that this sensor is working - meaning that only dissolved oxygen triggers the electrical change in the sensor, and not other atoms? and how would you calibrate the sensor to translate changes in electric signal to the change in number of dissolved oxygen atoms?

also, how would one get pure dissolved oxygen in varying amounts as 'positive control' for the sensor?

To start, the figure and explanation in the link are almost comically misleading. Potassium chloride solution does not "attract" oxygen. The oxygen flowing through the wire is pretty silly and

Where the probe joins the wire, oxygen mingles with the electricity. Oxygen is not very ionized, meaning that it does not have a negative charge as electricity does, so the oxygen dilutes the current at the electrode beyond the probe.

is just plain ridiculous. This one is better.

The way this type of probe works is by amperometric reduction of $\ce{O2}$, the first example being the Clark electrode. Essentially, an electrolyte is separated from the measurement solution by a porous film of poly(tetrafluoroethylene) (Teflon). These pores are small enough that only small substances like oxygen can diffuse through. On the electrolyte side, there are two electrodes in one of two possible configurations:

1. In the original Clark-type electrode, the working electrode is platinum, which is chemically-inert but catalyzes the reduction of oxygen. The counter electrode is a AgCl-coated Ag electrode that acts as a reference for the measurement. (Its potential is supposed to remain constant) To reduce the oxygen, an external potential must be applied across the two electrodes (normally using a potentiostat).
2. The galvanic cell–type electrode has one electrode made of silver (where the oxygen reduction occurs) and one made of an easily oxidized metal like lead. It works the same way as the Clark electrode, but instead of applying an external potential, the current required to reduce the oxygen is supplied by the oxidation of the lead electrode.

Any oxygen that reaches the working electrode is reduced and produces a current that is measured to produce the analytical signal. The amount of current produced depends on how much oxygen diffuses through the membrane, which depends on the concentration of dissolved oxygen in the test solution.

how would you know that this sensor is working - meaning that only dissolved oxygen triggers the electrical change in the sensor, and not other atoms?

This one is a bit tricky. You're right that oxygen isn't the only thing that could produce a signal—amperometry isn't a very selective technique on its own. You can control the redox reactions that occur at the working electrode, to a limited degree, by changing the potential you apply, i.e. different substances require different potentials to be reduced and won't be reduced if an insufficient potential is applied. In the case of this specific type of electrode, selectivity comes mainly from the PTFE membrane. Since it limits diffusion to only a few very small molecules, that greatly increases the selectivity of the electrode, though depending on the solution you're trying to measure, there still may be interferences.

and how would you calibrate the sensor to translate changes in electric signal to the change in number of dissolved oxygen atoms?

In principle, you could calculate the number of reduction reactions happening at the surface from Faraday's laws, but to turn this into a measure of the exact concentration in solution requires a lot of thought of how the transport of oxygen to the electrode occurs, from diffusion from the electrolyte to the electrode surface and transport from the test solution into the electrolyte. In practice, the assumption that the measured current is directly proportional to the concentration in solution is made (and is valid under many conditions), and a calibration is made using a standard solution.

also, how would one get pure dissolved oxygen in varying amounts as 'positive control' for the sensor?

Calibration solutions seem to be commonly made using tables of oxygen saturation at different temperatures. It's also possible to measure dissolved oxygen through other means, such as titration. Solutions can be made up, titrated, then the value used to calibrate an electrode.

Now, for the measurement specifically in cell cultures, a membrane electrode may not be the best bet, at least not without sample pre-treatment. The PTFE membrane is quite susceptible to adsorption of proteins and the like, which will probably affect the electrode's response. It may be necessary to somehow remove these contaminants (without affecting the dissolved oxygen concentration). Other options that might be viable are colourimetric measurements or titration, though there are problems with these, as well.