Oroboros Instruments O2k User manual

Oroboros Instruments High-resolution respirometry
Oroboros O2k-Manual
Mitochondrial Physiology Network 15.03(08):1-26 (2018)
Version 08: 2018-01-25 ©2010-2018 Oroboros
Updates: http://wiki.oroboros.at/index.php/MiPNet15.03_O2k-MultiSensor-ISE
O2k-MultiSensor
system with
ion selective
electrodes (ISE)
Fasching M, Gnaiger E
Oroboros Instruments
High-Resolution Respirometry
Schöpfstrasse 18, A-6020 Innsbruck, Austria
Email: [e-mail]
www.oroboros.at
Section Page
1. Introduction and scope 2
2. The ion selective electrode (ISE) system 3
2.1. The Oroboros O2k-TPP+ISE-Module
3
2.2. Assembly of the ISE 4
2.3. TPP+membrane conditioning and storage 7
2.4. Wash the ISE 8
2.5. Reference electrode: assembly, storage and maintenance 9
3. O2k-MultiSensor system 10
4. Operating instructions 11
4.1. Insert the ISE 11
4.2. Volume calibration with ISE-MultiSensor stoppers 12
4.3. Experiment 13
4.4. Instrumental background oxygen flux 14
4.5. ISE-calibration and performance test 15
4.6. Performance criteria 17
4.7. Troubleshooting 17
4.8. Membrane lifetime 18
5. O2k-MultiSensor control and calibration 18
5.1. pX signal 18
5.2. Configuration and gain 19
5.3. Calibration 20
Supplement A: DatLab 5.2. 22
Determine the O2k series 24
O2k series B and C, pX upgrade installed before 2011 24
O2k series B and C, pX upgrade installed after 2010 26

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1. Introduction and scope
The pX channel of the Oroboros O2k
yields a record of a potentiometric
(voltage) signal simultaneously with
the oxygen signal in both O2k-
chambers. The O2k-TPP+ISE-
Module consists of two ion-selective
electrodes (ISE) and separate
reference electrodes. The ISE can be
applied ff various hydrophobic
cations (TPP+, TPMP+), and other
cations (Ca2+, Mg2+), with
exchangeable membranes and
electrolyte. This manual describes
the application of the ISE system for
TPP+.
Left: O2k-MultiSensor with two
ISE inserted and TIP2k on top.
ISE The potentiometric channels are used with the ISE or
with an ion selective combination electrode (ISCE,
combining reference and measuring electrode in one
sensor body). The most common ISCE is the glass pH
electrode.
pX Potentiometric measurements result in a voltage signal
which is typically a linear function of the logarithm of
the activity (concentration) of the substance of interest
(the analyte). A calibrated pH electrode displays the
negative decadic logarithm of the H+ion activity
(potentia hydrogenii) and thus got its name “pH
electrode”. By analogy, an ISE may be used to
measure pTPP, pCa, etc., hence the general term “pX”
is used to denote the signal of an ISE.
Amp The O2k-FluoRespirometer not only includes the two
potentiometric channels, but two additional
amperometric (Amp; current) channels for optical
fluorescence sensors or amperometric sensors (NO,
H2O2, H2S).

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2. The ion-selective electrode (ISE) system
2.1. The Oroboros O2k-TPP+ISE-Module
ISE-Service Box, containing:
(1)
2Stopper\black PEEK\angular Shaft\side+6.2+2.6 mm Port, for application with ISE;
with 4 spare Viton O-rings (12x1 mm), with volume calibration ring
(2)
2Oroboros Ion-Selective Electrode TPP+and Ca2+:6 mm diameter shaft
(3)
ISE-Membrane Seal (spare)
(3)
ISE-Compressible Tube (spare)
(3)
4ISE-TPP+Membranes, PVC, 4 mm diameter, box of 5 membranes
(4)
ISE-Membrane Mounting Tool
(5)
Forceps for membrane application
(6)
ISE-Filling Syringe with needle
(7)
Stopper-Needle: Short needle for bubble extrusion from port of the ISE-stopper
(8)
2Reference-Electrode\2.4 mm: 2.4 mm diameter glass shaft, for ISE
(9)
4Replacement-Barrel for Reference-Electrode\2.4 mm
(10)
Electrolyte\Reference-Electrode
Manual O2k-MultiSensor System with ISE
For O2k Series B+C with pX upgrade installed before 2011 only
(11)
MultiSensor-Connector for separate reference electrode
(12)
Grounding cable with Allen key

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2.2. Assembly of the ISE
The ISE is delivered in an assembled state but without filling solution or
membrane. Before its first use it must be disassembled.
A ISE-Membrane Holder, lower part of electrode housing
BISE-Electrode Holder, middle part of electrode housing
CISE-Cable Connection, upper part of electrode housing
DISE-TPP+Membrane, each shipped between 2 paper disks
EISE-Membrane Seal
FISE-Compressible Tube
GISE-Inner Glass Electrode, with Ag/AgCl- and Pt-wire
HISE-Membrane Mounting Tool
2.2.1. Disassembly of the ISE
1. Unscrew part Bfrom part A
2. Insert the narrow end of
the ISE-Mounting Tool H
from the electrode tip into
part A (slightly angular)
and push the ISE-
Membrane Seal E,
compressible tube Fand (if
the electrode was already
in use) membrane Dout of
the housing.
Since no membrane is
mounted in a new ISE,
parts E+Fmay just slip out
of part A. In any case place
parts Eand Fimmediately
to a safe place (the black Cover-Slip may be used) to
avoid losing them.
3. Pull out the ISE-Inner Glass Electrode Gfrom the
housing B.
4. Unscrew part Bfrom part C.

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2.2.2. Membrane mounting
Use a good light source. Dry
all plastic parts (especially
the inside of parts A, E, and
F) with a paper towel.
1. With the forceps take a
membrane Dfrom the
membrane box and remove
the paper covers on both
sides of the membrane.
2. Place the membrane on the concave, broad side of
mounting tool H.
3. Holding tool Hwith the membrane upright, slide
housing part Acarefully over the tool (no old
membrane must have remained in
part A).
4. Insert tool Hwith the attached
membrane further into part A, holding
both parts upright. You may control
the progress by placing a good light
source behind part Aand viewing the
assembly against it. In this way you
will be able to see the movements of
the membrane and the tool inside part
A. If the membrane gets stuck to the
wall of part Acontinue to gently introduce it using
cycling movements to keep it straight. It is acceptable
if during part of the insertion process the membrane is
not flat on the tool. However, when you approach the
electrode tip make sure that the membrane is in a flat
position on the tool. Push tool Hwith the membrane
against the opening on the tip of the electrode, reverse
the orientation of the electrode (the tip now facing
down) and remove the tool gently while checking that
the membrane stays on the tip of the electrode housing
A.
5. Attach the ISE-Membrane Seal Eto the flexible ISE-
Compressible Tube F.
6. Insert the assembled parts E+F with the membrane
seal Efacing downwards to the membrane into
membrane holder A, with the electrode tip facing
downwards. Usually the assembled parts E+F will glide
downwards into the membrane holder A, otherwise
push it down with the flat end of mounting tool H.

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7. Finally press E+F gently against membrane D with the
flat end of mounting tool H.
8. Check that membrane Dis pressed flat (not folded)
against the opening in the electrode holder Aby
inspecting it against a light source.
9. Place the assembly (A+D+E+F) aside.
2.2.3. Fill the ISE-Inner Glass Electrode
Analyte
Membrane
Filling solution = Conditioning solution 1
TPP+
ISE-TPP+
10 mM TPPCl, 100 mM KCl
TPPCl Tetraphenylphosphonium chloride Sigma-Aldrich 218790
KCl Sigma-Aldrich 31248
Note: All TPPCl solutions described in this manual (filling,
conditioning, storage, calibration) can be stored at room temperature
in dark glass bottles.
1. Attach the ISE-Filling Syringe to the filling
needle and rinse the syringe once with the
filling solution.
2. Insert the needle as deep as possible into
the ISE-Inner Glass Electrode and slowly
fill the glass tube avoiding trapping of
bubbles.
3. The glass tube should be filled almost up
to its rim, leaving 1-2 mm empty to keep
the rim dry.
2.2.4. Final ISE assembly
1. Insert the ISE-Inner Glass Electrode G with the
platinum wire pointing down into cable connection C
pushing the platinum wire into the socket of part C.
3. Slide the electrode holder B over the ISE-Inner Glass
Electrode and partially (a few turns) screw it onto part
C. One thread on part B fits into membrane holder A,
the other thread to electrode connection C.
4. Hold the assembly of
(A+D+E+F) in one hand
and the assembly of
(G+B+C) in the other
hand, both need to be
horizontal. Then insert the ISE-Inner Glass Electrode G
into part A. Screw part A tightly onto part B.
5. Hold the entire assembly vertically with the electrode
tip upwards and slowly screw part C further into part B
while observing the formation of a bulb of the TPP+
membrane at the electrode tip.

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6. The bulb should protrude noticeable
from the electrode tip. The size of the
bulb can be controlled by screwing
part C more or less into part B. A good
result is usually obtained by fully
inserting part C into part B. However,
when the bulb starts to develop an
excessive size, reverse the tightening
and leave part C partially unscrewed.
7. To move the air bubble situated in the
tip of the electrode to the rear end of
the inner glass electrode, shake the
electrode like an (old fashioned, non-
electronic) fever thermometer: Point
the tip away from you and give the
entire electrode two or three short,
powerful shakes.
8. Compare the appearance of the membrane bulb before
and after shaking, noticing the difference between an
air-filled and a liquid-filled membrane bulb.
2.3. TPP+membrane conditioning and storage
Prior to use, the ISE must be conditioned. The first
stage of conditioning is performed in a solution
identical to the inner filling solution, see above.
Fill a 15 mL Falcon tube at least 1 cm high
with conditioning solution 1 and insert the
ISE with the electrode tip pointing
downwards into the solution. The conical
bottom of the tube prevents the membrane
bulb from touching the tube (this will NOT
work with a 50 mL Falcon tube). Allow at
least 24 hours of conditioning.
In the next step the electrode should be
conditioned in the storage solution. The
storage solution is equivalent to conditioning
solution 2.
The storage solution should contain the
same ionic background as the inner filling solution (and
conditioning solution 1 plus a concentration of the
analyte slightly lower than the desired experimental
range of measurement. Alternatively, no analyte and
just a solution maintaining the ionic background may
be used. The ISE may also be stored without liquid in
wet air, though this has not been tested for the
Oroboros system.
Bulb too big
Bulb ok

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Before inserting the ISE into the storage solution,
rinse the tip of the electrode with deionized water to
wash off traces of conditioning solution 1.
Analyte
Membrane
Storage solution = conditioning solution 2
TPP+
ISE-TPP+
1 µM TPPCl, 100 mM KCl
or 100 mM KCl
We recommend conditioning in the storage solution for
48 hours prior to first use of a newly mounted
membrane, although 24 hours may be sufficient for
many membranes. Some electrodes might reach their
full performance only in the second run after a new
membrane was mounted. Store protected from light.
2.4. Wash the ISE
The ISE has to be washed between experiments,
particularly if hydrophobic inhibitors and uncouplers are
used. The PVC membranes of the ISE are generally
only suitable for operation in aqueous media and are
damaged by non-aqueous solvents. Therefore, the
necessary washing steps between experiments have to
be carefully optimized according to specific
experimental regimes, and only some general
guidelines can be summarized here.
1. Remove the ISE from the stopper. Then the stoppers
can be washed separately in the O2k-chamber, using
the standard washing procedure (MiPNet19.03).
After carefully rinsing the ISE with deionized
water, rinse it with EtOH (do not immerse), and again
with plenty of water. Allow for re-equilibration in
storage solution. A long re-equilibration is preferable
(over-night), although electrodes have been used
successfully after only short re-equilibration times
(minutes). Test if this washing procedure is sufficient
for your experimental conditions, i.e. if carry-over of
inhibitors or uncouplers cannot be detected in the next
experiment.
2. A very effective cleaning procedure is immersion of the
electrode in a solution of living or dead cells (surplus
from cell cultures) or tissue homogenates in the O2k-
chamber. If necessary, this should be performed after
rinsing with (1) water, (2) ethanol, and (3) water.
3. In exceptional cases, it is necessary to immerse the
electrode in pure ethanol. In this case, check the
performance of the electrode by a calibration run
before relying on the electrode in any further
experiment. If the electrode does no longer or

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insufficiently respond to the analyte (even after re-
conditioning in the storage solution) the membrane
must be exchanged.
2.5. Reference electrode: assembly, storage and maintenance
The Reference-Electrode\2.4 mm for ISE is composed
of an internal silver-silver chloride electrode with an
internal filling solution of 3 M KCl saturated with AgCl.
Before the electrode can be put into operation, the
glass reference barrel must be filled with the
Electrolyte supplied for the Reference-Electrode.
Fill the reference barrel:
1. Unscrewing the white plastic cap of the reference
electrode: Remove the upper part of the cap with the
attached silver wire. Pull the glass barrel out of the
lower part of the cap.
2. The electrolyte solution is added to the glass tube using
the provided electrolyte bottle and polyethylene
tubing: Insert filling tube into nipple of electrolyte
bottle. Push until tube locks into place. Insert tube into
reference barrel and squeeze bottle. Fill reference
barrel up to approximately 0.5 cm (approx. 0.2 inch)
from top.
3. After filling the glass barrel with the reference
electrolyte, the silver wire is inserted into the glass
tube and the electrode cap is re-assembled.
Clean the electrode: To wash the reference electrode between
runs, rinsing is recommended in the sequence water,
pure ethanol, and water. This procedure should be
usually sufficient to prevent carry-over even of
hydrophobic inhibitors, since the reference electrode is
made of non-hydrophobic materials. Immersion into
pure ethanol should be avoided to prevent blocking of
the ceramic diaphragm in an assembled electrode.
When using the electrode in solutions containing higher
concentrations of protein, the electrode could be
soaked in a dedicated enzyme cleaning solution or a
chromic/sulfuric acid glass cleaning solution after each
use for 10-15 seconds to remove the protein from the
glass and the reference junction. This prolongs the
lifetime of the electrode.
Store the electrode: Always clean the electrode before
storage. Protect reference electrodes from light during
storage, e.g. by wrapping them in aluminum paper.

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Short term: Place the tip of the electrode in a test
tube or beaker containing reference electrolyte (3 M
KCl). Falcon type 15 mL vials are well suited. If
necessary, refill electrolyte before use.
Long-term (>4 weeks): Remove the glass barrel
containing the electrolyte and store the entire glass
barrel in a closed test tube filled with the reference
electrolyte. Rinse the silver wire and electrode cap to
remove the salt solution and dry using an absorbent
towel. Store in the accessory box or any closed
container to keep dust off the electrode and protect
from light.
Troubleshooting: Try to locate the problem either at the measuring
ISE or at the reference electrode by switching
electrodes. If you have only one reference electrode
you can switch to a spare glass barrel for diagnostic
purposes. The following text assumes that the problem
was located on the reference electrode.
Little or no response: Inspect the electrode for visible cracks.
If any exists, the glass barrel is defective and must be
replaced with a spare. The slightest crack in or around
the tip of the electrode may cause the electrode to read
about the same signal in all solutions.
Response pegs OFF scale: 1) Check the pX gain setting.
2) Visually inspect the electrode for broken or
dissolving internal Ag-AgCl wire or for inadequate
volume of reference electrolyte. Reference electrolyte
level should be above the Ag-AgCl element.
3) Blocked or clogged liquid junction –first clean
electrode tip, then soak it in warm (not hot) distilled
water for 5 to 10 min. If still clogged, remove the wire
from the glass barrel, clean the barrel with distilled
water, then soak it in distilled water. Next, clean it with
enzyme cleaning solution such as Terg-a-zyme
(Alconox, Inc.) to remove protein from the reference
junction. If still clogged, replace reference barrel with
spare barrel supplied.
3. O2k-MultiSensor system
The O2k-FluoRespirometer supports all add-on O2k-
Modules and includes all O2k-MultiSensor channels
mentioned below. For O2k- Series B and C see
Appendix.
Before handling the BNC plugs (on the O2k-Main
Unit) and connecting the electrodes, always touch the