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| The OROBOROS [http://www.oroboros.at/index.php?id=o2k-multisensor ISE system] is designed with replaceable membranes so it is possible to measure different ions like TPP+ or Ca<sup>2+</sup> with the same electrode housing.
| | #REDIRECT [[O2k-TPP+ ISE-Module]] |
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| A Ca<sup>2+</sup> selective membrane with a composition similar to the one described by Simon and coworkers <ref>Schefer U, Ammann D, Pretsch E, Oesch U, Simon W (1986) Neutral carrier based Ca<sup>2+</sup>-selective electrode with detection limit in the sub-nanomolar range. Analytical Chemistry 58: 2282-2285.</ref> has been tested with the Oroboros ISE system and detection limits of at least 10 nM were achieved in Ca<sup>2+</sup> buffered calibration solutions. This membrane is obtainable from Oroboros Instruments on request. There may be further optimizations of the membrane composition to ideally fit the the typical applications of Oroboros customers. However, the main obstacle in establishing useful Ca<sup>2+</sup> measurements appears to be not the Ca<sup>2+</sup> sensitive membrane but the methodology of its application in a medium simulating intracellular conditions.
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| ''Note: The operation of a Ca<sup>2+</sup> electrode at typically high extracellular Ca<sup>2+</sup> concentrations should be straightforward and devoid of the difficulties described here.''
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| These difficulties include both the calibration of the Ca<sup>2+</sup> electrode and the planning of a sensible biological experiment to use it. This is ongoing development and therefore Oroboros Instruments offers Ca<sup>2+</sup> selective membranes without scientific support beyond the information on this page, '''specifically to customers interested in developing a suitable protocol'''. While this support may grow in the future it will need user innovation and cooperation of users in the World Wide MiPNet to raise this support to a similar level as achieved for respiratory measurements!
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| ==Operation of the Ca<sup>2+</sup> electrode==
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| ===General===
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| The general operation (assembly, ...) of the Oroboros ISE system is described in [http://www.oroboros.at/index.php?id=o2k-multisensor MiPNet15.03].
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| While working with the pX channel please always observe the guidelines for avoiding damage to the electronics by [[ESD]].
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| ===Calculation of free Ca<sup>2+</sup> concentrations===
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| To work at physiological Ca<sup>2+</sup> concentrations requires usually Ca<sup>2+</sup> buffering by chelating agents. The calculation of c(Ca<sup>2+</sup>) free in such solutions is quite complicated and the results depend i.a on ionic strength, temperature, and -very strongly- on the solution pH value. Tools for such calculations are available on-line at Chris Pattons MAXCHELATOR Page [http://maxchelator.stanford.edu/ maxchelator.stanford.edu]. Many calculations, published or implemented in programs, depend on a single set of data compiled by Martell and Smith <ref>Martell AE, Smith RM (1989) Critical Stability Constants, 1: Amino Acids. Plenum Press</ref>.
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| Please note that in spite of the availability of easy to use software tools the calculation of free Ca<sup>2+</sup> concentration is an extremely difficult and - so to speak- daring task. The accuracy of these calculations is severely limited by several factors:
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| * many underlying thermodynamic constant may not be known precisely
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| * some additional thermodynamic constants necessary for adequate temperature correction may not be known at all
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| * it may be difficult to set the experimental parameters pH, temperature, and ionic strength with the necessary precision
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| ===Inner filling solution===
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| The following inner filling solution was used:
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| CaCl2: 10 mM
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| EDTA: 50 mM
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| pH adjusted to 8.5 with KOH
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| ===Conditioning===
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| is a controversial topic. If any conditioning is done, the used free Ca<sup>2+</sup> concentration should probable not be much higher than the highest expected concentration during use of the electrode.
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| ===Calibration===
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| As a potentiometer method the Ca<sup>2+</sup> electrode will deliver a signal that is (for the working range) linear to the logarithm of the free Ca<sup>2+</sup> concentration. Therefore, the electrode is calibrated by plotting electrode signal vs. logarithm of the free Ca<sup>2+</sup> concentration. Calibration of the Ca<sup>2+</sup> electrode at low (< 1µM) Ca<sup>2+</sup> levels is typically done by exposing the electrode to a series of Ca<sup>2+</sup> calibration buffers. Each calibration solution typically contains a Ca<sup>2+</sup> chelating agent, a pH buffer, CaCl2, and a salt (KCl) to adjust ionic strength. For suggested compositions see the literature in the reading list below. Examples from the literature may then be adapted to individual needs using the software tools mentioned above. The pH of all solutions have to be adjusted very carefully! For calibrations spanning a large (several orders of magnitude) range of free Ca<sup>2+</sup> concentrations it will be necessary to use calibration buffers with quite different compositions. The non linearity of the electrode response seen in such calibrations may be mainly due to the difficulties in calculating the free Ca<sup>2+</sup> concentration.
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| ==Use in a biological experiment==
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| You could help us in the development by explaining what you would like to do/ see in a Ca<sup>2+</sup> experiment and what you expect from the electrode: what ca concentrations do you want to measure, Ca<sup>2+</sup> release or uptake? what total concentration change in a 2 ml chamber (!) do you expect? Do you want to measure in a Ca buffered medium? (decreased sensitivity to changes) or without Ca<sup>2+</sup> buffering but then how do you get to physiological Ca<sup>2+</sup> concentration? Do you want to measure at physiological Ca<sup>2+</sup> concentrations?.......
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| Please use the [[Talk:Calcium electrode|Discussion page]] to leave your comments!
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| [http://www.bioblast.at/index.php?title=Special:UserLogin&type=signup Click here to create an account]
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| ==Reading list==
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| We have complied a short reading list that may be of interest to those planing to to do Ca<sup>2+</sup> measurements. A special emphasis are references describing the preparation of Ca<sup>2+</sup> calibration buffers.
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| [[Media:Ca_reading_list.pdf|Ca measurement reading list]]
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| ==References==
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| <references/>
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| <br>
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| {{#set:Technical service=Ca}}
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| <br> {{Technical service}}
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