1 Department of Systems Biology, Technical University of Denmark2 Agricultural and Environmental Proteomics, Department of Systems Biology, Technical University of Denmark3 Department of Micro- and Nanotechnology, Technical University of Denmark4 Bioanalytics, Department of Micro- and Nanotechnology, Technical University of Denmark5 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark6 Fluidic Array Systems and Technology, Department of Micro- and Nanotechnology, Technical University of Denmark7 Center for Intelligent Drug Delivery and Sensing Using Microcontainers and Nanomechanics, Center, 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 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.