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• W176247244 abstract "In this work, the P. putida respiration inhibition test was successfully transferred into the microplate format. Microplates with embedded fluorescent sensors warrant a high throughput and high sensitivity. Although intensity-based, the fluorescence signals showed excellent accuracy and reproducibility due to internal referencing using an analyte-inert reference dye. This makes the assay independent of well-to-well variations in film thickness or fluctuations in the excitation light intensity or the sensitivity of the detector and enables a calibration-free application of the set-up. Cross-sensitivities towards turbidity or fluorescent sample ingredients were excluded by optical isolation (pH sensor) and choice of a rather long-wave oxygen indicator. Concerning oxygen detection, the well-known complications resulting from oxygen ingress into the sample was investigated using various plate sealings and shaking speeds. The experiments were confirmed by mathematical simulations and the oxygen ingress roughly compared via kLa fits. Whereas the use of paraffin oil sealings cause fast oxygen ingress due to convection, rigid sealings were found to be quite effective considering the prevention of oxygen ingress. However, homogenous application of these sealings is rather complicated and cannot be automated. Small gas phases remaining between cover and sample lead to outliers which have to be sorted out manually, making evaluation rather time-consuming. Therefore, 150 µL of paraffin oil in combination with a low plate acceleration of the MTP in the reader were chosen for the screening tests due to much better well-to-well reproducibility and repeatability.Effects of the oxygen ingress on oxygen-consuming reactions were illustrated using an enzyme kinetic. For low enzyme concentrations and rather permeable plate sealings like paraffin oil, the oxygen content does not converge towards zero, but a steady-state is formed between oxygen ingress and consumption. The level of this steady-state depends on the enzyme activity. Here, enzyme activities cannot be obtained as the initial slope of the kinetic because oxygen ingress partially compensates the consumption, leading to incorrect kinetic parameters. However, formation of a steady-state can be used for endpoint evaluations. The properties of the sensor proved to be vital for the obtained results: Using relatively thick sensor layers of high oxygen solubility, the response time of the sensor is increased considerably because the sensor serves as an oxygen reservoir which releases oxygen into the sample. This results in too slow kinetics and corrupted kinetic parameters.Toxicological tests using the oxygen-sensitive MTP were investigated with respect to the reproducibility and accordance to a comparative experiment using a closed system. The assay was optimised with respect to the storage conditions of the bacteria solution, the MTP sealing and bacteria concentration. The resulting calculated inhibitions using 100 µL of paraffin oil as plate sealing combined with a low plate acceleration were constant over several hours and in good accordance with the comparative experiment and with values given in literature. The toxicological test was further performed with pH-sensitive MTPs and optimised considering the test solution ingredients and bacteria concentrations. Here, addition of 100 mM of NaCl and a higher bacteria concentration than used for the oxygen experiments was found to be vital, although shifting the dose-response curves towards higher inhibitor concentrations and therefore making the test less sensitive. The dose-response curves agreed very well with the ones detected with the oxygen measurements recorded with the same composition of test solution and bacteria concentration.Dose-response curves for different inhibitors were recorded with the oxygen- and the pH-sensitive MTPs. The resulting dose-response curves match rather well. This makes both the pH- and the oxygen-sensitive MTP an appropriate alternative to the oxygen electrode which enables high throughput screening of a large number of samples." @default.
• W176247244 created "2016-06-24" @default.
• W176247244 creator A5059379485 @default.
• W176247244 date "2006-03-14" @default.
• W176247244 modified "2023-09-27" @default.
• W176247244 title "Microrespirometry with sensor-equipped microtiterplates" @default.
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