3. O XYGEN I NGRESS INTO M ICROTITERPLATES AND ITS E FFECT ON K INETIC
3.4. C ONCLUSION
Detecting low enzymatic or metabolic rates based on oxygen measurements in MTP wells, oxygen diffusion through the cover and the MTP material play an important role and have therefore to be taken into account to obtain correct results. The cover should be preferably impermeable towards oxygen to detect even low oxygen uptake rates. For application in high-throughput screening, another parameter of high importance is the well-to-well reproducibility without any outliers which is the pre-requisition for automated evaluation.
Using MTPs, paraffin oil proved to be a poor protection from oxygen diffusion into the sample even with large amounts of paraffin oil, due to convection caused by inevitable movement of the MTP during the measurement. Shaking during lag times, which is sometimes necessary for homogeneity in the sample and prevention from precipitation of solid particles, increases the convection depending on the shaking mode. To avoid convection, rigid sealings have to be used. Foils of low or no permeability towards oxygen, e.g. based on PET or aluminium, would be the optimal protection from oxygen transfer through the cover if bubble-free sealing was possible.
However, an inevitable gas phase, which contributes largely to the oxygen reservoir in the sample if filling takes place under ambient air, the requirement of a maximum filling volume to minimise this gas phase, and cumbersome handling make these sealings less attractive. Paraffin wax dissolved in volatile solvents, which can be pipetted on the sample and gets almost rigid very quickly, avoids convection and a remaining gas phase and results in good well-to-well reproducibility but proved to be inapplicable due to inevitable temperature effects, cumbersome handling and, most important, low reproducibility of the resulting oxygen ingress because evaporation of the solvent during storage causes varying solvent content. With cap mats punctured for elusion of excess air, the same problem as with foil sealings occurred: Inevitable gas phases remained in some wells. These sorting out of outliers makes evaluation unacceptably labour-intensive for high throughput screening. The method of choice was the use of 100 µL paraffin oil using the lowest possible plate acceleration of the reader. Despite higher oxygen ingress than with rigid sealing, this method showed
Chapter 3: Oxygen Ingress into Microtiterplates and its Effect on Kinetic Parameters
Although suitable for the task of this work, our sealing of choice is not ideal for detection of lower oxygen consumptions due to the unavoidable convection of liquid plate sealings in MTP reader measurements, which can even lead to a total compensation of low OURs by oxygen ingress. For the application of rigid sealings is difficult to automatise, which is a major prerequisite for a high throughput, convection has to be decreased by other means. Choosing an MTP format with wells of a lower diameter, e.g. a 384-well or PCR plate, convection of the sample, especially covered with a liquid of high density, could solve this problem [27]. A contrary approach is the plate format designed by Papkovsky et al. [28]: The wells display a rather large surface-to-volume ratio. The MTP is covered with a thin glass lid as impermeable sealing, and excess sample is driven out into overspill areas. The interaction of the large aqueous surface and the glass lid provides for its adhesion on the sample. The samples themselves are kept in the wells by surface tension. However, besides the danger of cross-contamination due to overspilling, this format uses capillary effects and is designed for very small sample volumes (< 1 µL) and a dissolved oxygen indicator and is not applicable for coated MTPs.
Last but not least, convection can be eliminated by using a set-up where the MTP is stagnant and either the optical system moves for measurement, or the each well is addressed separately (c.f. SDR2, chapter 2.4.3). Both methods, however, complicate the instrument set-up considerably.
Aside from the oxygen diffusion through the cover, diffusion through the MTP material was observed. Furthermore, the PS MTP proved to act as an air reservoir which rapidly releases oxygen into samples with low pO2. This diffusion is negligible compared to the oxygen flux involving convection, but greater than the oxygen ingress from the cover using rigid sealings of lower permeability such as aluminium and PET foils or paraffin wax. Model simulations showed that this oxygen diffusion through the MTP material could be dramatically reduced using less permeable materials like PET. Another approach is the coating of the MTP with even less permeable polymer films made of PAN. Here, the latter method could be applied without the need of fabrication of completely new MTPs.
Chapter 3: Oxygen Ingress into Microtiterplates and its Effect on Kinetic Parameters
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Chapter 4: Pseudomonas Putida Respiration Inhibition Test