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

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O2k-Housing and follow the other procedures outlined
in (MiPNet14.01) to protect the O2k electronics from
damage by ESD.
Connect: Insert the plug of the ISE into the BNC plug labelled
“pX” on the front of the O2k-Main Unit, and the plug of
the reference electrode into the 2 mm pin plug labelled
“Ref” (MiPNet19.18A).
Gain: The gain of the pX channel is selected in the DatLab
software (Section 5.2). For measurements with the
Oroboros TPP+system, a gain of 20 is suggested.
4. Operating instructions
4.1. Insert the ISE
O2k-MultiSensor vs. standard stoppers: The introduction of
several (large) electrodes into the O2k-Chamber
through the stopper requires the use of “ISE-
MultiSensor stoppers”. The standard O2k-Stopper has a
concave shape on its end inserted into the chamber,
with a single capillary (gas-escape/titration capillary) in
the centre of the stopper (the highest point when
inserted). The end of the ISE-MultiSensor stopper is
angular with one capillary and two electrode inlets. The
gas-escape/titration capillary is at the side of the
stopper at the highest point when inserted.
Prevent bubbles: When inserting the stopper into the O2k-
Chamber filled with aqueous medium, gas bubbles are
guided into the gas-escape/titration capillary and
pushed out of the chamber. This is more effective,
however, with the standard stopper than the ISE-
MultiSensor stopper. Therefore, great care should be
taken to avoid the trapping of bubbles during initial
insertion. The single most critical point for prevention
of bubble formation is to close the chamber only after
full thermal equilibrium has been established. The best
criterion for thermal equilibrium is a stable oxygen
signal, with a slope near zero in the “open chamber”
configuration used for oxygen sensor calibration
(MiPNet19.18D).
1. Stop stirrers and fill the chamber with medium (2.35 mL for
a 2 mL chamber). Place the stoppers on top of the
chambers but do not yet close them. Activate stirring.
A gas phase like the one for air calibration has to be
visible. Using DatLab Graph layout “02a
TPP_calibration”, wait until temperature, Peltier power,

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and oxygen concentration are stable and the slope of
oxygen concentration is near zero (±1 pmol∙s-1∙mL-1).
2. Calibrate the oxygen signal (air calibration)
(MiPNet19.18D).
3. Stop the stirrers, and insert the stoppers completely
into the chambers.
4. Insert the ISE electrode into the larger (6 mm) ISE
inlet of the stopper. If a gas bubble remains in the
chamber (but liquid is on top of the stopper) try to
remove the gas bubble: inserting a short needle (flat
tip) without an attached syringe into the small titration
inlet usually removes any bubbles from the inlet,
thereby allowing the big bubble to escape from the
chamber. Smaller bubbles may be brought nearer to
the gas-escape capillary by starting and stopping the
stirrer several times. It may be necessary to lift the
entire stopper (including ISE electrode) to a position
above the liquid phase and insert it again.
5. Make sure that the smaller inlet for the reference
electrode (2 mm) is totally filled with liquid –if
necessary add more pre-warmed medium to the top of
the stopper.
6. Insert the reference electrode into the chamber. Move
it up and down to get rid of any bubbles that might be
trapped in its inlet. Switch on the stirrer and check for
any bubbles. If there are bubbles, repeat the
instructions described above.
7. Connect the electrodes to their plugs (Section 3).
8. Aspirate all excess liquid from the top of the stopper,
making sure the top is dry and no liquid film connects
the different inlets. The uncorrected slope of the
oxygen concentration should now be in the usual range
for a closed chamber at atmospheric saturation (2 - 4
pmol∙s-1∙mL-1). Considerably different fluxes may
indicate that there is a liquid “bridge” on top of the
stopper connecting at least two different inlets,
allowing the circulation of liquid between the chamber
and the top of the stopper.
4.2. Volume calibration with ISE-MultiSensor stoppers
When using an ISE-MultiSensor stopper, the ISE and
reference electrodes must be in place when calibrating
the O2k-chamber volume, comparable to volume-
calibration with standard stoppers (MiPNet19.18A).

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1. Add to the dry O2k-Chamber containing the stirrer bar
a water volume accounting for the final chamber
volume (2 mL) plus the additional dead volume in the
capillary and spaces between electrodes and inlets. For
the Oroboros ISE Assembly (ion selective electrode and
reference electrode), this additional volume is
approximately 0.16 mL. Therefore, the volume to
calibrate a chamber volume of 2 mL with the Oroboros
ISE system is 2.16 mL.
2. Prepare the ISE-MultiSensor Stopper (loose the
calibration ring, dry the stopper), making sure that the
three inlets are dry. Remove the ISE and the reference
electrode from their respective storage solutions. Dry
their shafts with a paper towel (do not use a paper
towel directly on the PVC membrane of the ISE or the
diaphragm of the reference electrode). Insert the
electrodes into the ISE-MultiSensor stopper.
3. Place the stopper on top of the chamber with a
loosened volume-calibration ring slid down to the
chamber holder. Insert the ISE-MultiSensor Stopper
slowly into the unstirred chamber carefully observing
first the diminishing gas phase in the chamber. Then
focus on the top of the stopper. Stop the insertion as
soon as the first drop of liquid appears on the top of
the stopper. This may be visible first on top of the gas-
ejection capillary comparable to the standard stoppers,
but it may also occur at the edge of the reference
electrode or the ISE.
4. Fix the position of the volume calibration ring by
tightening the screw as in the procedure with a
standard stopper.
4.3. Experiment
Two problems must be avoided while running an
experiment with an ISE- MultiSensor Stopper:
(a) Introduction of bubbles: After the chamber was filled as
described (Section 4.2), no gas bubbles should be
either in the chamber or in the capillary.
(b) Circulation of liquid between the top of the stopper and
the internal chamber needs to be prevented by
aspirating any excess liquid form the top of the
stopper. These conditions have to be maintained during
the entire experiment, removing excess liquid from the
stopper after any titration.
Injections: Before inserting a syringe needle into the stopper
(manual or TIP2k syringe), make sure that the capillary

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is filled with liquid –if necessary, place a drop of liquid
on top of the capillary - then remove any bubbles from
the capillary by using a needle without an attached
syringe. A gas-escape/titration capillary filled with
liquid without any gas bubbles provides good visibility
through the capillary to the light within the chamber. If
you cannot see the light, the capillary is blocked by gas
bubbles. These need to be removed. Similarly, when
the stirrer is switched off, an internally trapped gas
bubble might move into a position to block the light,
which can be checked further by switching the stirrer
on and off.
Insert the needle and perform the titration
(manual or TIP2k). After removing the needle,
remember to aspirate any excess liquid from the top of
the stopper that has been ejected from the constant-
volume chamber during titration. It is important to
minimize the time span during which a liquid bridge
exists between the different inlets through the stopper.
4.4. Instrumental background oxygen flux
Instrumental oxygen background parameters are used
to correct real-time oxygen flux (MiPNet14.06).
Instrumental background tests must be carried out
with the ISE-MultiSensor Stopper and all electrodes in
place. Instrumental background parameters obtained
with standard stoppers cannot be used for ISE-
MultiSensor experiments.
4.4.1. Dithionite background
Because of difficulties involved in opening and closing
the O2k-Chamber with an ISE-MultiSensor Stopper, it
is strongly recommended to use the instrumental
background procedure based on dithionite injections
(MiPNet14.06) to avoid repeated opening and closing of
the O2k-Chamber. Prepare the O2k-Chambers and ISE
as described above (MiPNet14.06). To prevent potential
damage to the ISE membrane, prolonged exposure to
an excess of dithionite should be avoided. Therefore,
the automatic zero calibration at the end of the TIP2k
program “BG_feedback” should be avoided, or the
electrodes be cleaned immediately after the injection of
the excess dithionite (last line of the TIP2k program).
In the TIP setup “BG_feedback_ISE” this last
program line has been deleted.

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4.4.2. Instrumental background parameters for oxygen flux
An O2k-Chamber with an ISE-MultiSensor stopper has
a higher oxygen backdiffusion, a0, at zero oxygen
concentration, as compared with a standard stopper.
In a 2 mL chamber using the Oroboros ISE system in
MiR06 at 37 °C, with an oxygen regime from air
saturation to low oxygen, the backdiffusion parameter,
a°, typically ranges from -4 to -8 pmol∙s-1∙mL-1. If more
negative fluxes (< -10 pmol∙s-1∙mL-1) are detected in
the background experiment, this is a strong indication
that a liquid bridge exists on the top of the stopper.
This problem can be solved by simply aspirating any
excess liquid from the top of the stopper.
4.5. ISE-calibration and performance test
4.5.1. Linear calibration
The voltage recorded between an ISE and reference
electrode is ideally a logarithmic function of the analyte
activity. Non-linear behaviour is observed below a
threshold concentration or due to electrode drift. A
multiple-point calibration is performed, recording the
electrode signal as a function of logarithmic
concentration over a wide concentration range. The
parameters of a linear fit (slope and intercept) are then
used for display of the calibrated ISE-signal. When
ionic strength is nearly constant during calibration and
experiment, concentrations may be used directly
instead of activities. This condition is usually met in
media used in biological experiments. When test runs
are performed in other media, a calibration medium
with near-constant ionic strength has to be used, such
as a 100 mM KCl solution. The calibration runs should
be performed right before a biological experiment using
experimental medium. In the case of a TPP+electrode
being used to determine membrane potential, ideally
the biological sample is injected into the O2k-chamber
directly after TPP+calibration.
A typical ISE-calibration before a biological
experiment should cover a slightly wider concentration
range than the one expected to occur during the
experiment. While it is possible to use a two-point
calibration, it is suggested to use at least 4 points for
calibration, unless a smaller number has been shown to
be adequate for the given task by experience.
Calibrations can be easily done using the Oroboros
TIP2k.

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4.5.2. Calibration range
The experimental TPP+concentration should be above
the limit of detection and below the inhibitory
concentration (O2k-Procedure MiPNet14.05). Decide
on a concentration range and steps to be used for
calibration, e.g. 0.7 µM to 1.5 µM TPPCl, in 5 steps:
0.7, 0.9, 1.1, 1.3, and 1.5 µM, respectively. The
electrode should be allowed to stabilize at the lowest
calibration concentration. Alternatively, the chamber
may be filled with medium already containing a
minimum analyte concentration.
4.5.3. ISE-calibration solution
The ionic background of the solution should be close to
the experimental medium. The best option is to use
experimental medium directly. When working with
MiR06 as a medium, 100 mM KCl solution is sufficient
for TPP+calibration, thus reducing the use of the more
viscose MiR06 medium, particularly with TIP2k
syringes. The analyte concentration in the calibration
solution should allow for injection volumes small
enough not to create major disturbances, but large
enough to allow for precise injections. In our example
a 100 mM KCl solution containing either 0.1 or 1 mM
TPPCl present good choices when using the TIP2k
(which allows precise handling of very small volumes).
When the calibration is performed by manual
injections, a 0.1 mM solution is used.
4.5.4. TPP+calibration with the TIP2k
Fill the O2k-chambers with medium and close the
chamber with electrodes inserted as described above.
Fill the TIP syringes with the calibration solution and
insert the TIP needles into the chambers.
Initial concentration: Use calibration solution 1 mM TPPCl in
100 mM KCl. A first injection of 1.4 µl into the 2 mL
O2k-Chamber increases the chamber concentration by
0.7 µM TPP+. This is performed with a TIP2k program:
Line
Mode
Delay
Volume
Flow
Interval
Cycles
S
µl
µl/s
S
1
D
1
1.4
40
1
1
Set the pX gain to 20 and allow the ISE-signal to
stabilize. The time derivative (slope) of the raw pX
signal (Section 5.3) should be in the range ±0.04 mV/s
. Drift is higher at extremely low (especially zero)
analyte concentration.

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TIP2k titrations: Start calibration titrations with the TIP2k
after a stable signal is obtained. The following TIP2k
setup can be applied, starting at 0.7 µM TPP+:
Why do you not simply give the name of the TiP2k setup
and simplify the description?
Line
Mode
Delay
Volume
Flow
Interval
Cycles
S
µl
µl/s
S
1
D
300
0.4
40
300
4
The TIP2k program line increases the concentration
from 0.7 to 1.5 µM in 4 cycles at steps of 0.2 µM.
Siphon off excess liquid from the top of the stoppers
after each injection.
The initial and subsequent titrations can be
combined in one TIP2k setup, allowing for a sufficiently
long stabilization period in line 1:
Line
Mode
Delay
Volume
Flow
Interval
Cycles
S
µl
µl/s
S
1
D
10
1.4
40
600
1
2
D
300
0.4
40
300
4
If necessary, suspend the program in line 1 until
stability is obtained.
Note that TPP+concentrations indicated above do
not take into account dilution effects (replacement of
liquid from the chamber). Correct concentrations must
be inserted into the calculation of the linear calibration
fit.
More details:
To write or edit a TIP Setup program: » MiPNet12.10.
» http://www.bioblast.at/index.php/MiPNet12.10_TIP2k-manual
4.6. Performance criteria
Calibration of the ISE provides a performance test.
1. Signal obtained at a low concentration: The signal
depends on the electrode type, the concentration, the
medium, the temperature, and the pX gain (in DatLab).
At 37 °C, 1 µM TPPCl and a gain of 10, the voltage of
the Oroboros TPP+electrode in MiR06 should be below
(more negative) than -1.3 V. At zero TPP+the signal
should be below (more negative) than -1.5 V. For a
gain setting of 20 these values are doubled.
2. Linearity of the signal / log (conc.) regression in
the experimental concentration range: This can be
assessed by the corresponding plot, by the regression
parameter R2, and by the deviation of data points from
the regression (the residuals), see pX calibration
window in Supplement A.
4.7. Troubleshooting

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If the required performance criteria are not met, the
following steps should be tested:
1. Set the polarisation voltage of the OroboPOS to 0.
Observe any effects on the pX raw signal. A tiny
potential jump is acceptable. If a drift in the pX signal
is either increased or reduced by this test or an
extreme jump in the signal observed, the membrane of
the OroboPOS should be replaced. Reset the
polarisation voltage to 800 mV after the test.
2. Shake the electrode as described above to make sure
that no air bubble is trapped at the tip of the electrode.
3. Condition the electrode for a longer time in storage
solution.
4. Repeat the entire conditioning process, starting with
conditioning solution 1.
5. Replace the membrane.
4.8. Membrane lifetime
Under experimental conditions, the lifetime of a
membrane is primarily determined by exposure to
organic solvents or inhibitor accumulation in biological
experiments. These factors vary considerably in
different applications. A membrane should only be
replaced when the performance is no longer
satisfactory.
5. O2k-MultiSensor control and calibration
5.1. pX signal
Graphs can be constructed to include both, recorded oxygen
and pX, or several graphs can be added to display
oxygen and pX data separately. All graph settings can
be saved as user-defined layouts (MiPNet19.18C).

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Reference layouts: Four reference layouts for pX are provided
in DatLab [Layout / Reference layouts / O2 pX].
5.2. Configuration and gain
In the O2k configuration window the pX electrode is
entered for documentation.
The gain for the pX channel is set in the O2k control
window [F7], tab Potentiometric, pX to 10, 20, 40, or
80. The gain amplifies the “pX Raw Signal”. Gain 1
yields the same voltage [V] as measured with any
multimeter between reference electrode and ISE.

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5.3. Calibration
In the example of TPP+calibration with TiP2k, the
values of -6.155, -6.046, -5.959, -5.886, -5.824
correspond to LogTPP+concentrations 0.7, 0.9, 1.1,
1.3, 1,5∙10-6 M respectively. The concentrations were
used as mark names and Log of these concentrations
for concentration in the marks specifications.
The traces show raw signal (in [V], upper trace) and
calibrated signal (in Log[TPP+], bottom trace) of TPP+
electrode.
Calibration for different signal types: If a pX channel was
calibrated for a pH electrode, these values will initially
also be used to calculate the calibrated signal when the
pH electrode is exchanged for a TPP+electrode. Even
when observing only the raw (not the calibrated)
signal, the time derivative (Slope pX) will be calculated
from the calibrated signal, which might lead to
confusion when the time derivative is used to assess
signal stability.
When previous calibration settings are needed
later (e.g. the channel is now again used with a pH
electrode), the old calibration values can be restored by
using the Copy from file button in the calibration
window and selection of the the file in which the