Non-Plastic: HRRHigh-Resolution Respirometry versus Multiwell System
The OROBOROS Oxygraph-2k for high-resolution respirometry (HRR) is the gold standard for highly accurate quantitative measurements, using very low amounts of sample. Instrumental and methodological approaches require an adequately advanced level of Quality Assurance in research and clinical applications.
Is respirometry in a multiwell system quantitative?
From their basic design, some multiwell systems appear to be a nice tool for qualitative high-throughput screening, for pharmacologic testing of a large number of substances. Designed for high-throughput qualitative measurements, they should not be advertised for quantitative measurements. Reliable quantitative results cannot be obtained with the present technology offered and advertised. Huge oxygen fluxes reported by one group at a mitochondrial physiology meeting were explained by a company representative as a "software problem". The absolute oxygen fluxes represent artefacts.
High-level instrumental specifications are required to achieve accurate quantitative results as obtained with the OROBOROS O2k: O2k-Concept.
Is plastic material compatible with respirometry?
Oxygen storage in the plastic material of multiwell plates leads to high oxygen backdiffusion. Since the problems are well known, specifications should be provided on backdiffusion (not awailable for multiwell system), hence no corrections can be made for backdiffusion. In contrast, HRR not only minimizes the effect of backdiffusion by avoiding plastic materials, but additionally allows for automatic correction for instrumental background flux (see O2k-specifications).
Mixing by moving the sensor/injector part up and down a few times is inadequate.
Plastic materials are eliminated from the chamber in HRR, and well controlled stirring is provided in the O2k.
How is the chamber volume defined?
No information is provided on the accuracy of the chamber volume in a multiwell system. The actual concentrations of titrated substances, therefore, are not know, in contrast to HRR.
How are cell number or mitochondrial protein defined?
How many cells are actually in the closed compartment for measurement of respiration in a well, or which fraction of isolated mitochondria is outside versus inside the chamber? How can the recorded change in oxygen concentration be converted to respiration per million cells or per mg protein? Without solving these problems, no quantiative measurements of respiration are possible.
Are multiple substrate-uncouplier-inhibitor titrations possible?
The number of different substances that can be titrated into a well is limited, such that modern substrate-uncoupler-inhibitor titration (SUIT) protocols cannot be applied in multiwell systems.
How high are the running costs?
How many of the wells of a dischargeable plate can actually be used for independent measurements? Several wells are required for calibration, edge effects may eliminate the use of wells on the sides. What is the cost of a plate and the cost of a single measurement? A plate with sensors and injectors can be used once only. The initial costs and the running costs are extremely high (e.g. expensive dischargeable wells for single use only). By comparison, the running costs for the OROBOROS O2k are very very low.
The OROBOROS O2k is not suited for a high-throughput approach. However, investing the identical amount of money, a large number of O2k chambers provides a unique high-throughput HRR system for quantitative O2k-measurements at low running costs.
Are specifications comparable?
No specifications are given on sensitivity (lower limit of detection of oxygen; non-linearity and restricted linear range; detection limit of oxygen flux) in such multiwell systems. Some advertise that they are so much better than Clark-type electrodes, but no quantitative specifications are provided. There are detailed and unique specifications for the O2k.
pH measurement
No specifications are given on sensitivity [µpH/s]. The effects of buffers including the bicarbonate system are ignored. The rate of acidification is presented in pH units per time, ignoring the log scale of pH. What is the drift of the pH signal?. An oxygen flux of 50 pmol/(s ml) corresponds - at an assumed O2 flux to extracellular H+ flux ratio of 1:1 - to a pH change of about 86 µpH/s in a very weak buffer (2 mM).
