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-Open Support
- » 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 indicate a biological contamination.
- Flux values lower than 1.5 pmol∙s−1∙mL−1:
- Air bubbles in the closed chamber: switch on the illumination of the O2k and inspect the chamber through the front window. Remove any air bubbles.
- A large volume of medium collected in the receptacle of the stopper: siphon off excess medium.
- A larger chamber volume: check O2k-Chamber volume calibration.
- Quality control f: The zero signal at mark R0 for zero calibration (not shown) should be <2% of R1 (stable at <5% is acceptable).
Troubleshooting
General
- 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:
- USB-Cable 2.0\Type A-B not properly connected to the O2k and PC or Laptop.
- O2k-Chamber not properly positioned, such that O2 sensors are not connected to the medium.
- OroboPOS-Membranes defective or not properly applied.
- OroboPOS-Connector contaminated gold contacts; plugs not properly connected to the sockets of the O2k-Main Unit.
- OroboPOS contacts not cleaned. In rare cases, Pen-Contact Oil might be used.
- OroboPOS-Electrolyte Powder contaminated, inappropriate water used for dissolving the powder.
- O2-Zero Powder not properly handled; confused with OroboPOS-Polishing Powder.
- OroboPOS not properly serviced; not properly mounted to the OroboPOS-Connector; or defective POS head.
- Room temperature not sufficiently stable.
- Stirrer-Bar\white PVDF\15x6 mm not added to the chamber, or stuck and not rotating.
- O2k-Barometric Pressure Transducer not properly calibrated.
- O2k-Peltier Temperature Control defective electronics.
- O2k-Electromagnetic Stirrer Twin-Control defective electronics.
- 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.
- 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.
- Empty the chamber with the O2k running and connected to DatLab. Switch the stirrer off. Disassemble theOroboPOS from the OroboPOS-Connector as exemplified.» O2k-Videosupport: Disassembly of 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.
- 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.
- 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.
- For O2k-Series D and higher: The signal should be 0 V, otherwise an electronic problem of the O2k-Main Unit is indicated.
- 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 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.
Switch components approach
- If specifications given in the POS-SOP are not obtained: switch components for locating the problem.
Next step - O2k Quality Control 2: MiPNet14.06 Instrumental O2 background (Switch components troubleshoot approach to be provided as a Instrumental DL-Protocol within the new software)
- Fill the O2k-Chambers with medium or water and perform Oxygen_calibration_-_DatLab following the SOP as in MiPNet06.03_POS-calibration-SOP.
- When the O2 slope neg. signal is stable, empty the O2k-Chambers (keep recording!), mark the event (F4) and describe it in the 'Event' window.
- 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 OroboPOS-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 OroboPOS-Connector, prevent damage by electrostatic discharge by following the guidelines: MiPNet14.01 ESD-damage.
- Refill the O2k-Chambers with pre-warmed medium, mark and denote the Event (e.g., Sensors swapped).
- Repeat the O2-Sensor test as in 1).
- Repeat step 2).
- Switch the OroboPOS-Connector between O2k-Chambers A and B.
- Repeat step 4), mark and denote the Event (e.g. Connectors swapped). Please note that OroboPOS remain in the same chamber as before ( NO swap).
- Repeat the O2-Sensor test as in 1).
- Repeat step 2).
- Repeat the OroboPOS swap as in step 3), mark and denote the Event (e.g., Sensors swapped).
- Repeat the O2-Sensor test as in 1).
- Repeat the O2-Sensor test with your original setup, i.e., OroboPOS and OroboPOS-Connector in their original O2k-Chambers.
- Customer ID: DE_Seewiesen_Casagrande_S
Question: We have a problem located in the O2k-Chamber B, its OroboPOS is not providing the recommendation specifications of MiPNet06.03_POS-calibration-SOP. I am attaching an image to visualize the issue. I have performed the POS service 6 times including cathode and anode cleaning | O2k-Videosupport, OroboPOS-Holder exchange, O2k-Chamber disassembly followed by assembly, and stirrers exchange. Nevertheless, the signal is always very bad. The data is attached (2019-11-28).
Answer:
1. To discern the origin of your observed noisy signal - could you perform a switch approach by recording the |QC1: Oxygen sensor test within one measurement DLD file including switch of the OroboPOS between chamber A and B, 'ie.': O2k-Chamber A with OroboPOS A + OroboPOS-Connector A and O2k-Chamber B with OroboPOS B + OroboPOS-Connector B followed by O2k-Chamber A with OroboPOS B + OroboPOS-Connector B and O2k-Chamber B with OroboPOS A + OroboPOS-Connector A followed by O2k-Chamber A with OroboPOS B + OroboPOS-Connector A and O2k-Chamber B with OroboPOS A + OroboPOS-Connector B followed by O2k-Chamber A with OroboPOS A + OroboPOS-Connector B and O2k-Chamber B with OroboPOS B + OroboPOS-Connector A. 2. Perform a complete POS-service (including cathode cleaning, anode cleaning, dry the connector with a clean and dry tissue after the service) as explained in MiPNet06.03_POS-calibration-SOP. 3. Perform the |QC1: Oxygen sensor test after the complete POS-service and send me the resulting files for further evaluation.
Customer feedback: Following the quality control tests I have obtained a drastic signal improvement. The data is attached (2020-01-13).
Keywords: Oxygen signal
- Bioblast links: Oxygen signal - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Specific
- O2k-Procedures
- MiPNet O2k-Procedures
- General
- » Oxygen, dioxygen, O2
- » Oxygen calibration - DatLab
- » Oxygen solubility
- » Oxygen solubility factor
- » Oxygen pressure
- » Concentration
- » Activity
- » Pressure - Pascal
- » Barometric pressure
- » High-resolution respirometry
- » OroboPOS
- » Polarographic oxygen sensor
- » MitoFit Quality Control System
- » Sensitivity
- General
- Other keyword lists
MitoPedia O2k and high-resolution respirometry: O2k hardware, DatLab, Oroboros QM