O2k-TPP+ ISE-Module

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O2k-TPP+ ISE-Module

Description	O2k-TPP+ and Ca2+ ISE-Module: 2 potentiometric ion-selective electrodes (ISE), ISE-Service Kit and 2 PVDF-Stoppers, supported by the O2k-Core.
Product ID	12300-01
Type	O2k, O2k-Module, MultiSensor, Stopper, Catalogue
Link	O2k-Multisensor @OROBOROS, ISE-Manual, Tetraphenylphosphonium
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O2k-TPP+ and Ca2+ ISE-Module consists of:

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General

The OROBOROS ISE system is designed with replaceable membranes making it possible to measure different ions such as TPP⁺ or Ca²⁺ with the same electrode housing.

O2k-Core and O2k Series A-E

For the Oxygraph-2k Series A-E, the O2k-TPP⁺ and Ca²⁺ ISE-Module is supported by the O2k-Core and by all O2k instruments with the O2k-MultiSensor electronic upgrade.

TPP /TPMP electrode

The main purpose of the O2k-TPP+ and Ca2+ ISE-Module is to measure Mitochondrial membrane potential via the TPP or TPMP method.

The first publication using the O2k-TPP+ and Ca2+ ISE-Module for this purpose was Brown 2011 Am J Physiol Regul Integr Comp Physiol

Calcium electrode

We recommend to use fluorescence methods for measuring Ca²⁺ concentrations, utilizing the O2k-Fluorescence LED2-Module. Fluorescence based methods for detection of Ca²⁺ are more easy to set up and are more widely used in biosciences tan ISE based methods. However, there may be special applications in which determination of Ca²⁺ levels via ISE is advantageous. Also a laboratory already using the O2k-TPP+_and_Ca2+_ISE-Module but without funds for the O2k-Fluorescence LED2-Module may want to use ISE based Ca²⁺ detection. For this purposes ISE-Ca2+ Membranes suitable for use with the O2k-TPP+_and_Ca2+_ISE-Module can be ordered from OROBOROS instruments and are described here:

A Ca²⁺ selective membrane with a composition similar to the one described by Simon and coworkers has been tested with the OROBOROS ISE system and detection limits of less than 10 nM were achieved in Ca²⁺ buffered calibration solutions. 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 Ca²⁺ measurements appears to be not the Ca²⁺ sensitive membrane but the methodology of its application in a medium simulating intracellular conditions.

Note: The operation of a Ca²⁺ electrode at typically high extracellular Ca²⁺ concentrations is straightforward and devoid of the difficulties described here. These difficulties include both the calibration of the Ca²⁺ electrode and the planning of a biological experiment to use it.

ISE-Ca2+ Membranes for use in the Oroboros ISE system can be ordered separately.

Operation of the Ca²⁺ electrode

General

The general operation (assembly, ...) of the Oroboros ISE system is described in MiPNet15.03.

While working with the pX channel please always observe the guidelines for avoiding damage to the electronics by ESD.

Calculation of free Ca²⁺ concentrations

To work at physiological Ca²⁺ concentrations requires usually Ca²⁺ buffering by chelating agents. The calculation of c(Ca²⁺)_free in such solutions is quite complicated and the results depend i.a on ionic strength, temperature, and - very strongly - on the pH. Tools for such calculations are available on-line at Chris Pattons MAXCHELATOR Page maxchelator.stanford.edu. Many calculations, published or implemented in programs, depend on a single set of data compiled by Martell and Smith . Note that in spite of the availability of easy to use software tools the calculation of free Ca²⁺ concentration is not simple. The accuracy of these calculations is severely limited by several factors:

• Many underlying thermodynamic constants may not be known precisely
• Some thermodynamic constants necessary for adequate temperature correction may not be known at all
• It may be difficult to set the experimental parameters pH and ionic strength with the necessary precision

Inner filling solution

The following inner filling solution is used: CaCl₂: 10 mM

EDTA: 50 mM

pH adjusted to 8.5 with KOH

Conditioning

Conditioning of the membrane is a controversial topic. If any conditioning is done, the used free Ca²⁺ concentration should probable not be much higher than the highest expected concentration during applications of the electrode.

Calibration

As a potentiometric method, the Ca²⁺ electrode delivers a signal that is (in the working range) linear to the logarithm of the free Ca²⁺ concentration. Therefore, the electrode is calibrated by plotting electrode signal vs. logarithm of the free Ca²⁺ concentration. Calibration of the Ca²⁺ electrode at low (< 1µM) Ca²⁺ levels is typically done by exposing the electrode to a series of Ca²⁺ calibration buffers. Each calibration solution typically contains a Ca²⁺ 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 range (several orders of magnitude) of free Ca²⁺ concentrations it is necessary to use calibration buffers with very different compositions. The nonlinearity of the electrode response in such calibrations may be mainly due to the difficulties in calculating the free Ca²⁺ concentration.

Application in biological experiments

You could help us in the development by explaining what you would like to do/ see in a Ca²⁺ experiment and what you expect from the electrode: what ca concentrations do you want to measure, Ca²⁺ 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 Ca²⁺ buffering but then how do you get to physiological Ca²⁺ concentration? Do you want to measure at physiological Ca²⁺ concentrations?.......

• Please add your comments in the Discussion page. Pease contact [email protected] to recive an account.

• user feedback: FCCP may be incompatibe with the function of the ISE-Ca2+ Membranes. Fasching Mario 11:45, 2 November 2012 (CET)

Reading list

We have complied a short reading list that may be of interest to those planing to to do Ca²⁺ measurements, with special emphasis on references describing the preparation of Ca²⁺ calibration buffers.

Ca measurement reading list

References

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