Zentrum Angewandte Chemie · 2015. 7. 30. · Zentrum Angewandte Chemie Institut für Technische...
Transcript of Zentrum Angewandte Chemie · 2015. 7. 30. · Zentrum Angewandte Chemie Institut für Technische...
Zentrum Angewandte ChemieInstitut für Technische ChemieCallinstr. 5 , D-30167 Hannover
Andreas Prediger1, Tim Höpfner , Patrick Lindner , Mehriban Akin ,
Merve Yuksel , Sascha Beutel , Suna Timur , Thomas Scheper
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1Leibniz University Hannover, , Callinstr. 5, D-30167 HannoverInstitute of Technical Chemistry
A multifunctional at-line analysis systemfor monitoring of cell cultivations
Control software
An integration of further sensors into the system is desired. A fibre optical detection of thepH-value could be carried out analogously to the pO -value detection. The used enzymes
in the biosensor could be exchanged to allow the detection of other relevant biologicalsubstances such as lactate or organic acids.New image processing algorithms are constantly developed. An aim for the future is thecell viability determination via optical properties of the cell without the need to stain them.One approach for this is the implementation of neuronal networks.A new flow microscope prototype for fluorescence measurements is under development.It will be used for at-line monitoring of micro algae cultivations and could be integrated intothe multisensor system as well.
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Funded by
2Ege University, Faculty of Science, Biochemistry Department, 35100 Bornova-Izmir, Turkey
Perspectives
Fig.3: Monitoring of a cultivationS.cerevisiae
Image processing
Fig.1: Schematic setup of the multisensor system
Biosensor
IntroductionIn biotechnology lots of interesting products are produced in suspension cellcultures. Process security and a high quality of the final product are essentialin this field. This is why continuous monitoring of the bioprocesses is essential.Amultifunctional at-line analysis system was developed to allow the automaticacquisition of process data from different sensor systems. Fig.1 shows asimplified schematic setup of the device.A flow microscope (Fig.2) allows the monitoring of cell density and cell viability.Dead cells are stained using dyes (e.g. Methylen blue) and the acquiredimages are analyzed with an image processing software. The developementof glucose and ethanol concentrations during a cultivation can be monitoredusing a dual biosensor. The pO -value is determined via fibreoptical
measurements.A mixing chamber that is attached to an injection system allows the automaticdilution of samples and thus the monitoring of broad cell density andconcentration ranges.
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Fig.2: Flow Microscope1
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1: Connection for control and power supply2: CCD camera3: Micrometer screws4: Microscope body5: Connections for flow cell6: Illumination system with connection cable
Fig.4: Original image
Fig.5: Result imageof software
The acquired images are analyzedwith the processing software "
.Using an edge-detection algorithmthe cells in the image are detectedvia grey value gradients.Subsequently the mean grey value ofthe cell is determined. If it exceeds adefined threshold the cells aremarked as living (yellow box). If it isbelow the threshold the cells aremarked as dead (blue circle).
Life
and Death"
Five living cells and onedead cell are detected
by the image processingsoftware
Aflow electrode with a dual biosensor is used for the detection of glucose andethanol concentrations.Pyranoseoxidase und ethanoloxidase are immobilized separately on twogold electrodes using PAMAM (Polyamidoamine). They catalyze thefollowing reactions:
Glucose + O Gluconolactone + H O
Ethanol + O Acetaldehyde + H O
The substrate concentration is measured indirectly via the oxygenconsumption. Oxygen is detected at a potential of -700 mV and thus nointerference with media ingredients occurs.The linear range of both biosensors is between 0 and 0.5 mM. A stability testshowed that after 11h 98% (Pyranoseoxidase) and 94% (Ethanoloxidase) ofthe initial enzyme activity were retained.
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in the WTZ Programme
Published in: Akin . 2011 A new set up for multi-analyte sensing: At-line bio-process monitoring.Biosensors & Bioelectronics 26:4532-4537.
et al .
Fig. 3 shows the result from monitoring of an example cultivation ofwith the multisensor system. After calibration all data were
acquired completely automatically.S.cerevisiae
The device is controlled with the software FIA-Master. Via a RS232 serialinterface all pumps, valves and the illumination system can be controlled. Allactions are determined with a script that is easily created and modified fordifferent applications.