Oxygen sensor test

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Oxygen sensor test

Description

The O2 sensor test is an important component of the MitoFit Quality Control System. The OroboPOS sensor test is described in detail in MiPNet06.03 POS-calibration-SOP, is performed after switching on the Oroboros O2k, and is required as a basis of technical service of the instrument.

Abbreviation: POS test

Reference: MiPNet06.03 POS-calibration-SOP





MitoPedia O2k and high-resolution respirometry: O2k hardware, DatLab, Oroboros QM 



O2k-Open Support
This information is part of O2k-Open Support.


O2 sensor test
» 1. Service and preparation
» 1a. O2k-Manual: POS-service
» 1b. O2k-Protocols SOP: O2k-cleaning
» 2. POS-calibration SOP
» 2a. Stirrer test
» 2b. O2k-Manual: O2k-calibration

Figure 2. POS Quality control using the DatLab protocol (MiPNet06.03 POS-calibration-SOP)

1. Even before final equilibration, perform a stirrer test [F9], switching both stirrers automatically off an on.
2. About 20 min are required for approximate air equilibration after temperature equilibration of the incubation medium, visualized as stabilization of the Peltier power(Fig. 2; time scale is 1:10h:min).
Quality control a: Upon automatic re-start of the stirrer (On), the increase of the oxygen signal should be rapid and monoexponential.
Quality control b: The raw signal (blue plot; 1 V = 1 μA at gain 1)should be close to 1 to 3V at 25 to 37 °C at sea level up to 1,000 m altitude, in the range of pb 101 to 90 kPa (at gain setting of 2 the raw signal [V] would betwo times higher).
3. Within 40 min, the oxygen signals should be stable with O2 slope (uncorrected) close to zero(Fig. 2).
Quality control c: Signal noise should be low, reflected in a noise of the O2 slope (red plot) within ±2 (±4 is acceptable) pmol∙s−1∙mL−1at a data recording interval of 2 s and 40 data points selected for calculation of the slope (Fig. 2).
4. Set a mark on the oxygen signal (R1) and click on O2 Calib. to open the DatLab O2calibration window.
Quality control d: The slope uncorrected should be within ±1 pmol∙s−1∙mL−1averaged across the section of the experiment marked as R1 for air calibration(d).The recorded POS signal should be close to the previous calibration under identical experimental conditions. See O2-Calibration window (Fig. 2; b’).
5. Continue with a complete instrumental O2background test(MiPNet14.06)or simply close the chamber and if required perform a zero oxygen calibration.
Quality control e: After closing the chamber, select plot Y2 and set mark J°1. Background slope (neg.) should be within 2.5±1 pmol∙s−1∙mL−1. Flux values higher than 4.0 pmol∙s−1∙mL−1 may indicate a biological contamination.
Quality control f: The zero signal at mark R0 for zero calibration (not shown) should be <2% of R1 (stable at <5% is acceptable).


Trouble shooting

The O2 sensor test does not only serve to evaluate the function of the OroboPOS, but many other components of the O2k have to function according to specifications for a high-resolution oxygen signal to be obtained:
  1. USB-Cable 2.0\Type A-B not properly connected to the O2k and PC or Laptop.
  2. O2k-Chamber not properly positioned, such that O2 sensors are not connected to the medium.
  3. OroboPOS-Membranes defective or not properly applied.
  4. OroboPOS-Connector contaminated gold contacts; plugs not properly connected to the sockets of the O2k-Main Unit.
  5. OroboPOS contacts not cleaned. In rare cases, Pen-Contact Oil might be used.
  6. OroboPOS-Electrolyte Powder contaminated, inappropriate water used for dissolving the powder.
  7. O2-Zero Powder not properly handled; confused with OroboPOS-Polishing Powder.
  8. OroboPOS not properly serviced; not properly mounted to the OroboPOS-Connector; or defective POS head.
  9. Room temperature not sufficiently stable.
  10. Stirrer-Bar\white PVDF\15x6 mm not added to the chamber, or stuck and not rotating.
  11. O2k-Barometric Pressure Transducer not properly calibrated.
  12. O2k-Peltier Temperature Control defective electronics.
  13. O2k-Electromagnetic Stirrer Twin-Control defective electronics.
  14. O2k-Main Basic not properly connected; other defective hardware.


If the signal remains off scale (9.99 V) or very low (< 1 V) at air saturation (25 to 37 °C; lower signals at lower temperatures): Many components may be responsible, and an electronic defect of the O2k-Main Unit must be carefully excluded.
  1. Check settings for Gain (use Gain 1) and Polarization voltage (use 800 mV). If these settings were incorrect, the off-scale problem may be solved simply by using the standard settings.
  2. Empty the chamber with the O2k running and connected to DatLab. Switch the stirrer off. Remove the OroboPOS-Connector with the attached OroboPOS. Leave the sensor attached to the POS connector, and the POS connector plugged into the O2k-Main Unit. Record the signal for some minutes. The raw signal should normalize to a value >1 V and <3 V (Gain 1). If so, the O2k-Chamber assembly was problematic (application problem), and re-assembly will solve the problem. » O2k-Videosupport: Insert O2k-Chamber.
  3. Remove the sensor head from the sensor connector, which remains plugged into the O2k-Main Unit. If the raw signal is not stable at 0 V, a defect of the OroboPOS-Connector is indicated.
  4. Remove the plug of the OroboPOS-Connector from the O2k-Main Unit. A signal of 0.4 V, which remains stable when changing the gain, is a strong indication of an electronic problem in the O2k-Main Unit.
    1. For O2k-Series D and higher: The signal should be 0 V, otherwise an electronic problem of the O2k-Main Unit is indicated.
    2. For O2k-Series A to C: The signal should be off-scale (+/- 9.99 V), otherwise an electronic problem of the O2k-Main Unit is indicated.
If specifications given in the POS-SOP are not obtained: switch components for locating the problem.
  1. Switch the OroboPOS between O2k-Chambers A and B. This is a good opportunity to clean the gold contacts and apply Contact oil to the gold pin and thread connecting the POS connectors and sensors. It is not necessary to remove the seal tip and the mounted membrane from the OroboPOS. When disconnecting a sensor from the POS connector, prevent damage by electrostatic discharge by following the guidelines: MiPNet14.01 ESD-damage.
  2. Repeat the O2 sensor test. If the problem moved together with the sensor from one side to the other, the problem is located in the OroboPOS sensor.
  3. If the problem remained on the same side, switch the POS connector together with the attached sensors between right and left side (A and B).
  4. Repeat the O2 sensor test. If the problem moved together with the POS connector, it is located at the OroboPOS-Connector.

O2k-QCS Next step - O2k Quality Control 2: MiPNet14.06 Instrumental O2 background

O2k open support cases

  • Customer ID: CH Lausanne Kayser B

Question:

I ran a zero oxygen calibration after the instrumental background protocol and the O2 neg slope for O2 concentrations of 60 and 30 uM is positive and greater compared with the O2 neg slope at O2 concentrations of 180 and 120 uM. The data is attached (2019-09-11).

Dith oxidized 2019-09-11 P1-05.DLD.png

Answer: Stepwise analysis of the attached data shows the following: Quality control (QC) 1: Your POS operates perfectly: during air calibration the O2 slope neg. approaches zero (see section 2.1 of https://wiki.oroboros.at/images/7/77/MiPNet06.03_POS-Calibration-SOP.pdf); Quality control (QC) 2: In the closed chamber, the O2 slope neg. is <4 pmol s-1 mL-1 which excludes any biological or chemical contamination from the MiR05 buffer. https://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf;

  • From your dithionite titrations we can observed that:
  1. After titrations of 2.5 µL of Dith, the step representative of O2 consumption is too small – indicative of a low effective potency of the solution of Dith;
  2. At increasing concentrations of Dith towards stepwise lower O2 concentrations, we observe that the O2 slope negative does not decrease in a titration dependent manner. Therefore, O2 is being consumed by the solution even after the initial fast decline of oxygen. Since we excluded other potential problems, the irregular background traces are due to the Dith solution.(see representative trace on page 5 of https://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf)

Taken together, your dithionite is highly oxidised and a continuing oxidation reaction explains the results.

Solution: Assuming you prepared the Dith according to our SOP (always fresh, http://bioblast.at/index.php/Dithionite), I advise you to purchase a new Dith powder (your Dith may be old and/or has oxidized).