Talk:O2k-Fluo LED2-Module

Measuring hydrogen peroxide

See Measuring H2O2 production with any brand similar to Amplex(R) REed see Amplex red and especially the accompanying Discussion page. For an overview of methods to mneasure H2O2 concentration or H2O2 production see Measuring hydrogen peroxide.

Simultaneous measurement of TPP and fluorescence

Question: Will it be possible to measure JO2, fluorescence AND TPP simultaneously, or just JO2/fluorescence vs JO2/TPP?

Answer: Background The main unknown is the chemical compatibility of the methods. We still have to find out whats the effect of Amplex red and its reaction product on the TPP signal. If this is not a big problem there are technical issues: a black TPP stopper will be required and then still light entering the chamber through the TPP electrode might be a problem. Producing the TPP electrodes in black is presumable not a very good idea because then it will no longer be possible to "see" the position of the membrane. I think the technical problems are solvable but first we have to check for chemical compatibility.

Update: First exploratory experiment:

A chamber was equipped with TPP electrodes (black stoppers) and a fluorescence module for measuring H2O2 via the Amplex Red method. A standard TPP calibration was carried out. Amplex red / HRP addition resulted only in a minor signal in the TPP channel, a further TPP calibration showed no obvious large negative effects on the performance of the TPP electrode. An H2O2 calibration of the fluorescence signal was carried out: The sensitivity for H2O2 was reduced by a factor of 3 to 4 as compared to measurements without TPP electrodes. At the same time the noise was drastically increased. In the comparative experiment (without TPP electrodes) no random noise was seen - digital resolution was the limiting factor. Based on this comparison, noise increased in the experiment with TPP electrodes at least by a factor of 20, probable more. It will be possible to estimate this factor with a higher precision after running an experiment without TPP electrodes at a higher gain (higher digital resolution).

It was further tested whether the presence of TPP+ alone (without electrodes) could explain this behavior but, if there were any effects at all, they were quite small.

Peroxide additions corresponding to a concentration change of 110 nM (110 pmol/ml) were still well visible but not additions corresponding to a 22 nM (22 pmol/ml) concentration change.

This was only one exploratory experiment (2 chambers)!

Based on this preliminary data: An important point is why one would want to measure TPP and H2O2 simultaneously in the same chamber: If this is "only" for time / sample economy the results above strongly discourage this idea. Parallel measurement e.g one chamber TPP / one chamber H2O2 are preferable. If there is a very strong need to measure both parameters on the same sample (e.g. if strong in-homogeneity among samples in this respect is suspected) and a comparable high H2O2 flux is expected the simultaneous measurement might be principally doable, especially if further optimizations are possible. E.g.: Maybe with the TPP electrode in the chamber black stirrers become important again? However, this certainly would involve some methodological work and the results would be second class at the best.

What would be your driving interest for simultaneous measurements?

best greetings Mario

Question: How many parameters can be measured at once?

Answer:

The O2k-system is very flexible. If you take cultured cells or isolated mitochondria, split them into the two chambers of the O2k, then you can maximize the number of simultaneously measured parameters:

In both chambers oxygen concentration and oxygen flux (O2k-Core; http://www.bioblast.at/index.php/O2k-Catalogue:_O2k-Core );