Dufva, Martin

Emnéus, Jenny

Finnie, Christine

Heiskanen, Arto

Mark, Christina

Svensson, Birte

Programmed cell death (PCD) in plants can influence the outcome of yield and quality of crops through its important role in seed germination and the defence process against pathogens. The main scope of the project is to apply microfluidic cell culture for the measurement of electrochemically or optically detectable events during PCD in barley aleurone layer, a cell model for living plant tissues, for a better understanding of the underlying mechanisms of PCD in plants. Microfluidic cell culture enables in vitro experiments to approach in vivo conditions. The major advantage of electrochemical sensors and detection systems is that they can be miniaturized, multiplexed and automated without losing their performance making them suitable for integration with microfluidic devices1,2. Combining microfluidics with electrochemical and optical detection allows implementation of a wide range of assays for online, real-time, parallel analysis of important parameters such as redox activity (NADPH:NADP ratio), H2O2 concentration, oxygen consumption, extracellular pH, cell viability and release of target enzymes (α-amylase and limit dextrinase). Probing the intracellular redox activity is of major importance, since it is known that reactive oxygen species, which are affected by changes in the redox activity of the cells3, are involved in PCD in plants, but the relationship between and mechanisms behind ROS and PCD is only poorly understood in plant cells4. Recently, it has been shown, using optical detection, that the H2O2 concentration changes depending on phytohormone activation or inactivation of aleurone layer metabolism and subsequent PCD3. Currently, we are working on the optimization of an intracellular whole-cell redox activity (NADP:NADPH ratio) assay5 to be able to detect possible changes of the cellular redox activity in barley aleurone layer. In our initial experiments using the electrochemical mediatorassisted assay we observed changes in the redox activity with tendencies similar to those for the H2O2 concentrations presented by Ishibashi et al. Further experiments are needed in order to improve reproducibility of the measurements and to find the optimal parameters suitable for its application in the microfluidic device. Meanwhile, we successfully detected PCD induced by phytohormones in barley aleurone layer using a double-fluorescent probe-system also used by Fath et al6, and it is planned to integrate this system in the microfluidic device.</p

Online Research Database In Technology

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Dec 19, 2017Monitoring programmed cell death of living plant tissues in microfluidics usingelectrochemical and optical techniquesMark, Christina; Heiskanen, Arto; Svensson, Birte; Emnéus, Jenny; Dufva, Martin; Finnie, ChristinePublication date:2012Document VersionPeer reviewed versionLink back to DTU OrbitCitation (APA):Mark, C., Heiskanen, A., Svensson, B., Emnéus, J., Dufva, M., & Finnie, C. (2012). Monitoring programmed celldeath of living plant tissues in microfluidics using  electrochemical and optical techniques. Abstract from 8thProtein.DTU Workshop, Kgs. Lyngby, Denmark.Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques Christina Mark1,2, Kinga Zór2, Arto Heiskanen2, Birte Svensson1, Jenny Emnéus2, Martin Dufva2 and Christine Finnie1 1Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark 2Department of Micro- and Nanotechnology, Technical University of Denmark  Brief description of research area:  Programmed cell death (PCD) in plants can influence the outcome of yield and quality of crops through its important role in seed germination and the defence process against pathogens. The main scope of the project is to apply microfluidic cell culture for the measurement of electrochemically or optically detectable events during PCD in barley aleurone layer, a model for living plant tissues, for a better understanding of the underlying mechanism of PCD in plants.  Microfluidic cell culture enables in vitro experiments to approach in vivo conditions. The major advantage of electrochemical sensors and detection systems is that they can be miniaturized, multiplexed and automated without losing their performance making them suitable for integration with microfluidic devices1,2. Combining microfluidics with electrochemical and optical detection allows implementation of a wide range of assays for online, real-time, parallel analysis of important parameters such as redox activity (NADPH:NADP ratio), H2O2 concentration, oxygen consumption, extracellular pH, cell viability and release of target enzymes (α-amylase and limit dextrinase).  What we know:  Probing the intracellular redox activity is of a major importance, since it is known that reactive oxygen species, which are affected by changes in the redox activity of the cells3, are involved in PCD in plants, but the relationship between and mechanisms behind ROS and PCD is only poorly understood in plant cells4. Recently, it has been shown, using optical detection, that the H2O2 concentration changes depending on phytohormone activation or inactivation of aleurone layer metabolism and subsequent PCD3. Currently, we are working on the optimization of an intracellular whole-cell redox activity (NADP:NADPH ratio) assay5 to be able to detect possible changes of the cellular redox activity in barley aleurone layer. In our initial experiments using the electrochemical mediator-assisted assay we observed changes in the redox activity with tendencies similar to those for the H2O2 concentrations presented by Ishibashi et al. Further experiments are needed in order to improve reproducibility of the measurements and to find the optimal parameters suitable for its application in the microfluidic device. Meanwhile, we successfully detected PCD induced by phytohormones in barley aleurone layer using a double-fluorescent probe-system also used by Fath et al6, and it is planned to integrate this system in the microfluidic device.   References 1 Xu et al., Talanta, 2009, 80, 8-18., 2009 2 Sassa et al, Electrophoresis, 2008, 29, 1787-1800, 2008 3 Ishibashi et al., Plant Physiol, 158, 1705-1714, 2012 4 Gechev et al., BioEssays, 28, 1091-1101, 2006 5 Heiskanen et al., Anal Biochem, 2009, 384, 11-19, 2009 6 Fath et al., Plant Physiol, 126, 156-166, 2001  Acknowledgements This project is funded by the Danish Agency for Science, Technology and Innovation. 

Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Dec 19, 2017Monitoring programmed cell death of living plant tissues in microfluidics usingelectrochemical and optical techniquesMark, Christina; Heiskanen, Arto; Svensson, Birte; Emnéus, Jenny; Dufva, Martin; Finnie, ChristinePublication date:2013Document VersionPeer reviewed versionLink back to DTU OrbitCitation (APA):Mark, C., Heiskanen, A., Svensson, B., Emnéus, J., Dufva, M., & Finnie, C. (2013). Monitoring programmed celldeath of living plant tissues in microfluidics using  electrochemical and optical techniques. Abstract from PlantBiotech Denmark Annual Meeting 2013, Frederiksberg, Denmark. Poster abstract   Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques  Christina Mark1,2, Kinga Zór2, Arto Heiskanen2, Birte Svensson1, Jenny Emnéus2, Martin Dufva2 and Christine Finnie1  1Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, 2Department of Micro- and Nanotechnology, Technical University of Denmark  Programmed cell death (PCD) in plants can influence the outcome of yield and quality of crops through its important role in seed germination and the defence process against pathogens. The main scope of the project is to apply microfluidic cell culture for the measurement of electrochemically or optically detectable events during PCD in barley aleurone layer, a cell model for living plant tissues, for a better understanding of the underlying mechanisms of PCD in plants. Microfluidic cell culture enables in vitro experiments to approach in vivo conditions. The major advantage of electrochemical sensors and detection systems is that they can be miniaturized, multiplexed and automated without losing their performance making them suitable for integration with microfluidic devices1,2. Combining microfluidics with electrochemical and optical detection allows implementation of a wide range of assays for online, real-time, parallel analysis of important parameters such as redox activity (NADPH:NADP ratio), H2O2 concentration, oxygen consumption, extracellular pH, cell viability and release of target enzymes (α-amylase and limit dextrinase). Probing the intracellular redox activity is of major importance, since it is known that reactive oxygen species, which are affected by changes in the redox activity of the cells3, are involved in PCD in plants, but the relationship between and mechanisms behind ROS and PCD is only poorly understood in plant cells4. Recently, it has been shown, using optical detection, that the H2O2 concentration changes depending on phytohormone activation or inactivation of aleurone layer metabolism and subsequent PCD3.  Currently, we are working on the optimization of an intracellular whole-cell redox activity (NADP:NADPH ratio) assay5 to be able to detect possible changes of the cellular redox activity in barley aleurone layer. In our initial experiments using the electrochemical mediator-assisted assay we observed changes in the redox activity with tendencies similar to those for the H2O2 concentrations presented by Ishibashi et al. Further experiments are needed in order to improve reproducibility of the measurements and to find the optimal parameters suitable for its application in the microfluidic device. Meanwhile, we successfully detected PCD induced by phytohormones in barley aleurone layer using a double-fluorescent probe-system also used by Fath et al6, and it is planned to integrate this system in the microfluidic device.   References 1 Xu et al., Talanta, 2009, 80, 8-18, 2009 2 Sassa et al, Electrophoresis, 2008, 29, 1787-1800, 2008 3 Ishibashi et al., Plant Physiol, 158, 1705-1714, 2012 4 Gechev et al., BioEssays, 28, 1091-1101, 2006 5 Heiskanen et al., Anal Biochem, 2009, 384, 11-19, 2009 6 Fath et al., Plant Physiol, 126, 156-166, 2001  Acknowledgements This project is funded by the Danish Agency for Science, Technology and Innovation. 

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Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques

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