Toward microbioreactor arrays : a slow-responding xxygen sensor for monitoring of microbial cultures in standard 96-well plates

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a 96-well plate was developed. The slow response time ensures that the measured oxygen value does not change much during plate transport to the microplate reader. The sensor therefore permits at-line DO measurement of microbial cultures. Moreover, it eliminates the necessity of individual optical measurement systems for each culture plate, as many plates can be measured successively. Combined with the 96-well format, this increases the experimental throughput enormously. The novel sensor plate (Slow OxoPlate) consists of fluorophores suspended in a polymer matrix that were placed into u-bottom 96-well plates. Response time was measured using sodium sulfite, and a t90 value of 9.7 min was recorded. For application, DO values were then measured in Escherichia coli and Saccharomyces cerevisiae cultures grown under fed-batch–like conditions. Depending on the DO sensor’s response time, different information on the oxygenation state of the culture plate was obtained: a fast sensor variant detects disturbance through sampling, whereas the slow sensor indicates oxygen limitation during incubation. A combination of the commercially available OxoPlate and the Slow OxoPlate enables operators of screening facilities to validate their cultivation procedures with regard to oxygen availability.BMBF, 02PJ1150, Plattformtechnologien für automatisierte Bioprozessentwicklung (AutoBio

Microrespirometry with sensor-equipped microtiterplates

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

Characterization of microtiterplates with integrated optical sensors for oxygen and pH, and their applications to enzyme activity screening, respirometry, and toxicological assays

This paper describes properties of microplates (MTPs) with integrated, fluorescence-based sensors for pH and oxygen, and their application for enzyme screening and monitoring of bacterial respiratory activity. Thin, hydrophilic sensing films consisting of an analyte-sensitive indicator and a reference fluorophore, are deposited on the bottom of the MTP. This allows for calibration-free quantification of pH and pO2 with an acceptable accuracy and resolution for applications such as enzyme activity screening, respirometry, or toxicological assays. The sensor properties are mainly investigated with respect to their qualification for such applications. Specifically, enzyme activity screening is demonstrated for glucose oxidase using oxygen-sensitive plates, and for bacterial growth monitoring of Escherichia coli and Pseudomonas putida. Furthermore, a toxicological assay monitoring the respiration activity of P. putida was converted into a microplate format

Cardiotoxicity testing using pluripotent stem cell‐derived human cardiomyocytes and state‐of‐the‐art bioanalytics: a review

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state‐of‐the‐art bioanalytical methods and sensors is reviewed. The focus is on hESC‐derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given.JRC.F.1-Health in Societ

Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: a review.

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given