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MiPNet15.03 O2k-MultiSensor-ISE

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MiPNet15.03 O2k-MultiSensor-ISE


Publications in the MiPMap
O2k-Manual
O2k-MultiSensor system with ion selective electrodes (ISE).

ยป Bioblast pdf - Versions

OROBOROS (2014-03-06) Mitochondr Physiol Network

Abstract: Fasching M, Gnaiger E (2014) O2k-MultiSensor system with ion selective electrodes (ISE). Mitochondr Physiol Network 15.03(05):1-20.

O2k-Manual: The ion selective electrode (ISE) is a modular O2k-MultiSensor extension of the OROBOROS O2k. ISE yield a potentiometric (voltage) signal simultaneously with the oxygen signal in both chambers of the O2k. The ISE system consists of separate reference and measuring electrodes selective for hydrophobic cations (TPP+, TPMP+), Ca2+, Mg2+, etc. This O2k-Manual describes the handling and application of the ISE system.

ยป Product: O2k-TPP+ ISE-Module, O2k-Catalogue

โ€ข Keywords: HRR, O2k-MultiSensor

โ€ข O2k-Network Lab: AT_Innsbruck_OROBOROS


Labels: MiParea: Instruments;methods 





HRR: Oxygraph-2k, TPP, O2k-Manual, O2k-Protocol 

O2k-MultiSensor, DatLab 



Template NextGen-O2k.jpg


MitoPedia O2k and high-resolution respirometry: O2k-Open Support 



O2k signal and output

  1. O2k signal: The O2k-TPP+ ISE-Module is operated through the pX channel of the O2k, with electric potential (volt [V]) as the primary and raw signal
  2. O2k output: type C

In Series D and higher the ISE connectors consist of 2 ports:

  1. A BNC port: both poles are "live" so it can be used either for a combination electrode (like a classical pH) or for just one singe electrode that requires a separate reference electrode.
  2. A 2 mm pin type connection that is used for connecting the separate reference electrodes for TPP and pH.


Long-term storage of the TPP electrode

For long-term storage (several months) disassemble the electrode, wash all parts with ethanol (sterilization), and allow all parts to dry completely. Then re-assemble the dry electrode without a membrane (avoid loosing the small parts). Store the electrode in the TPP accessory box (dark).

TPP electrode - interaction with TMPD

  • TMPD traveling into the TPP probe: pretty sure it was happening because the electrolyte was turning blue. And actually it became a persistent problem with several probes (at first I thought maybe there was a leak around the membrane off of one probe) (it would shift the absolute voltage values to more positive, until it the signal gets super staticky and I couldn't use the data at all). I resorted to either making sure I inverted the probe a few times to redistribute the TMPD in the electrolyte to spread it out a bit... or switching out the electrolyte (without replacing the membrane) and letting it sit overnight. And of course minimizing contact time of the probe with a solution containing TMPD.
~ Contribution by Gigi Lau from CA Vancouver Richards JG.


Technical service for the ISE system

Sometimes it is difficult to find out whether problems with the ISE system are caused by the electrodes (TPP, Ca, reference, pH) or by the pX electronics of the O2k. The following tests can help to solve this question.

Testing a TPP system outside the O2k-Chamber

This test allows to test for any disturbing influences in the chamber, like crosstalk to the OroboPOS. It is therefore similar to the test proposed in the manual (switching off the O2 polarization voltages) but has a broader scope (covering also e.g. possible interference from stirrers) and provides a more complete separation of POS an pX electrodes.

Place TPP electrode and reference electrode together in one falcon tube filled with electrolyte solution (e.g. media). Connect the cables of the electrodes to the O2k and record the pX signal. If a problem previously observed disappears in this test (and is reproducible by using the same electrodes in the O2k-Chamber) the problem is located in the chamber, e.g. at a leaky POS membrane, see the manual.


Testing a TPP system with a pH electrode (and vice versa)

This method allows to test the sensitivity of the pX electronics.

Requirements

Another type of electrode than can be connected to the ISE system. So if the problem is observed with an ISE system for measuring TPP, a pH electrode is the usual choice because it is available in many labs. The only requirements for the second electrode is that it can be connected via a BNC port. It does not have to be an electrode from OROBOROS Instruments neither is it necessary that the electrode fits into the O2k chamber. However, note that using a pH electrode outside the O2k-Chamber also constitutes a test similar to the one described above (Testing a TPP system outside the O2k-Chamber). Therefore a problem caused e.g. by a leaky POS membrane will not be observed when testing with a pH electrode outside the chamber.

Procedure

  • Connect the pH electrode to the oxygraph.
  • Put the electrode into a pH 4 calibration buffer (event)
  • Observe (record) the signal in DatLab.
  • Put the electrode into a pH 7 calibration buffer (event). Obviously the sequence of buffers does not matter.
  • From such a DatLab file we will see whether the pX electronics is working and even get a very rough estimation of the gain.

Testing TPP or pH electrodes with a voltmeter

This test measures the performance of the electrodes independently form the pX electronics of the O2k.

Requirements

Any voltmeter, frequently labeled "Multimeter" if different measurement modes can be selected, suitable for measuring mV potentials. .

Procedure for TPP electrodes

  • Set up the TPP /reference electrode in the O2k chamber as usually, only medium in the chamber.
  • Connect the electrodes to the pX electronics and record the signal.
  • Disconnect both electrode cables from the O2k (but leave the electrodes in the chamber, stirring still on).
  • If you have a "Multimeter" available set in into "Voltmeter mode"
  • Set the recording range of the voltmeter to a range good for recording about 200 mV
  • now measure the voltage difference between reference electrode and TPP electrode by touching (ideally fixing) the two probes of the voltmeter to the ends of the cables from the electrodes: for the reference electrodes this is simply the gold colored pin, for the TPP electrode this is the INNER , central pin. Note down the voltage reading on the voltmeter display. For this step the help of a second person is most convenient, otherwise you probable have to find a way to fix the cables in some way. It is not necessary to have the voltmeter in contact with the electrodes all the time, just when you are reading the values.
  • Increase the TPP concentration in a few (lets say 5) rather large steps (at least 2 ยตM per step) and note down the displayed values.
  • Finally reconnect the electrodes to the pX electronics of the O2k and observe how much (if at all) the signal has changed there. * Please send us the recorded values along with the TPP concentrations used and the the DatLab file recorded at the same time (that shows the readings for the first and last point). If no change in signal is visible with the voltmeter, the problem is at the TPP electrode, not the O2k.

Procedure for pH electrodes

  • Follow the procedure for TPP electrodes, moving the pH electrode between different pH calibration buffers instead of doing a TPP titration.

Ca measurement

ISE-Ca2+ Membranes are not included in the O2k-TPP+ ISE-Module but can be ordered from OROBOROS INSTRUMENTS separately (Product ID 42280-01), for application of the O2k-TPP+ ISE-Module for Ca2+ measurements.

The OROBOROS ion selective electrode (ISE) is designed with replaceable membranes making it possible to measure different ions such as Ca2+, TPP+, TPMP+ with the same electrode housing. See also Calcium for general consideration about Ca2+ measurements. We recommend to use fluorescence methods for measuring Ca2+ concentrations, utilizing the O2k-Fluo_LED2-Module. However, there may be special applications in which determination of Ca2+ levels via ISE is advantageous. The use of a Ca2+ electrode in mitochondrial research was described by Moreno et al.[1] Laboratories who are using the O2k-TPP+ ISE-Module (but who do not have the O2k-Fluo_LED2-Module) may apply the ISE for Ca2+ detection.

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 (< 1ยตM) Ca2+ levels is typically done by exposing the electrode to a series of Ca2+ calibration buffers. For a discussion of Ca2+ calibration buffers see Calcium#Ca2.2B_calibration.

Calculation of free Ca2+ concentrations:ยป Calcium#Calculation_of_free_Ca2.2B_concentrations

Ca2+ electrode and FCCP

There are strong indications that FCCP and the ISE-Ca2+ Membranes are incompatible.


Other ions beside TPP+ TPMP+, and Ca2+

There are ion selective membranes available, e.g. from Sigma Aldrich, for a wide variety of species. If cut to a circle with a diameter of 4 mm these membranes can be used in the ISE system. Many ions may also be detected by fluorimetric based methods, see O2k-Fluorescence_LED2-Module.


References

  1. โ†‘ Moreno AJM, Vicente JA (2012) Use of a calcium-sensitive electrode for studies on mitochondrial calcium transport. Methods Mol Biol 810:207-17.