MiPNet06.05 Test Experiments on O2k-Specifications

                

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MiPNet06.05 Test Experiments on O2k-Specifications

Gnaiger E (2010) Test experiments on specifications of the Oroboros Oxygraph. Mitochondr Physiol Network 6.5.

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Oroboros (2010) Mitochondr Physiol Network

Abstract: Summary: The Oroboros O2k with DatLab software is world-wide the only instrument on the market, which allows routine measurements to be made with specifications summarized under the term “high-resolution respirometry”: The limit of detection of O₂ flux is as low as 0.5 pmol.s^-1.mL^-1. Signal noise at zero oxygen concentration is <0.05 μM O₂. Oxygen backdiffusion at zero oxygen is <3 pmol.s^-1.mL^-1, and oxygen consumption at air saturation and standard barometric pressure (100 kPa) was 2.7 +/- 0.9 SD in 114 test runs at 37 °C. These highly standardized instrumental background fluxes are a linear function of oxygen concentration, which is used for routine background correction of oxygen flux. Typical exponential time constants of the oxygen sensors are <4 s, used for dynamic corrections in kinetic studies. Results of an extensive experimental test program are presented as the basis of Oroboros O2k-Specifications, which distinguish high-resolution respirometry from any conventional oxygraph system.

• O2k-Network Lab: AT Innsbruck Oroboros

O2k High-resolution respirometry

The Oroboros O2k with DatLab software is world-wide the only instrument (sole source) which allows routine measurements to be made with specifications summarized under the term high-resolution respirometry (HRR).

Continuous control of the Oroboros O2k-Specifications are a fundamental component of Quality Assurance and distinguish high-resolution respirometry from any conventional oxygraph system and from less quantitative approaches.

» O2k-Specifications

Oroboros O2k: technical specifications

General specifications

• Dimensions - O2k-Main Unit: L 45 cm, W 31 cm, H 25 cm; 13.45 kg without packing box or Peli Case.

• Chambers: chemically inert and minimum oxygen diffusion:

- Two Duran glass chambers with 2 mL (1.5 to 3.5 mL) volume; 16 mm inner diameter.

- PEEK stoppers with Viton O-rings.

- Electromagnetic stirrers with variable speed (100 to 900 rpm).

- PVDF-coated stirrer bars (6 mm diameter).

- Polarographic oxygen sensors sealed with butyl rubber seal tips.

• Built-in electronically regulated Peltier thermostat:

- Temperature range:

At room temperature	4 °C to 47 °C
At lower ambient temperature	2 °C

- Temperature stability:

Temperature stability

±0.002 °C over 90 min.

- Temperature range:

in 15 min	20 °C to 30 °C
in 20 min	30 °C to 20 °C

• Data output through USB:

- Two oxygen signals.

- Pressure transducer, for absolute barometric pressure signal, resolution 0.1 kPa.

- Thermostat temperature signal, resolution 0.001 °C.

- O2k-MultiSensor:

Series J and XB

• two additional amperometric signals (O2k-fluorescence; PhotoBiology)
• two additional potentiometric signals (Ion-selective electrodes: for TPP⁺, Ca²⁺, or pH)

Series XB

• two additional amperometric signals for Q redox state
• two additional amperometric signals (NO)

• DatLab software:

- Simultaneous display of O2 concentration and O2 flux (negative slope or time derivative), together with any selected O2k-MultiSensor channels (raw or calibrated signals and corresponding time derivatives).

- Barometric pressure for O2 calibration; temperature stability control.

- Automatic calibrations: O2 and O2k-MultiSensor channels.

- DL-Protocols: real-time guide through instrumental quality control tests and advanced diagnostic substrate-uncoupler-inhibitor titration (SUIT) protocols within a single assay for exploring mitochondrial function.

• internal PC and attached screen

Specifications for high-resolution respirometry

• Oxygen signal:

- Noise at zero O₂: <±0.02 µM (SD, 100 data points recorded at 0.2 s intervals at 37 °C) without smoothing (±0.005 µM typical)

- Noise at zero O₂: <±0.002 kPa (SD, 100 data points recorded at 0.2 s intervals) without smoothing (±0.0005 kPa typical).

- Noise at air saturation: <±0.1 µM O₂ (SD, 100 data points recorded 0.2 s intervals at 37 °C) without smoothing, at 180 µM O₂ (±0.05 µM typical).

- Noise at air saturation: <±0.01 kPa (SD, 100 data points recorded 0.2 s intervals) without smoothing, at partial oxygen pressure of 20 kPa (±0.005 kPa typical).

- O₂ range of linearity: 0 to 1000 µM.

- Time constant: <4 s at 37 °C (<3 s typical).

• Oxygen flux J_O2,V [pmol.s^-1.mL^-1]:

- Limit of detection: 0.5 at steady-state over 5 min.

- Sensitivity (normoxic): <1 at steady-state over 5 min at 20-40 °C.

- Sensitivity (hyperoxic): <3 at steady-state over 5 min at 20-40 °C.

- Noise: <0.2 after standard smoothing (120 s).

- O₂ range: flux measured up to 500 µM O₂ (permeabilized fibres), and <0.1 µM based on DatLab analysis of oxygen kinetics (mitochondria and cells).

• Instrumental background for linear correction over the entire oxygen range:

- O₂ backdiffusion [pmol.s^-1.mL^-1] at 0 µM: <3 at 20-40 °C (2.5 typical).

- O₂ backdiffusion [pmol.s^-1.mL^-1] at 0 kPa: <3 at 20-40 °C (2.5 typical).

- O₂ consumption [pmol.s^-1.mL^-1] at 200 µM: <4 at 37 °C (3 typical); <3 at 25 °C (2 typical).

- O₂ consumption [pmol.s^-1.mL^-1] at 20 kPa: <4 at 37 °C (3 typical); <3 at 25 °C (2 typical).

Labels: MiParea: Instruments;methods 

HRR: Oxygraph-2k