1 Department of Systems Biology, Technical University of Denmark2 Agricultural and Environmental Proteomics, Department of Systems Biology, Technical University of Denmark3 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark4 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
This project focuses on applying microfluidic tissue culture for electrochemical or optical measurements during programmed cell death (PCD) in barley aleurone layer to increase understanding of the underlying mechanisms of PCD in plants. Microfluidic tissue culture enables in vitro experiments to approach in vivo conditions. Microfluidics also allow implementation of a wide range of electrochemical or optical assays for online, real-time, parallel analysis of important parameters such as redox activity, O2 and H2O2 concentration, extracellular pH, cell viability and enzyme activity1,2. Currently, we are optimising an intracellular whole-cell redox activity assay3 that detects changes in redox activity in barley aleurone layer during PCD. The assay uses a double mediator-system to electrochemically measure redox activity via changes in the NADP:NADPH ratio. Initial experiments assay show that the redox activity changes depending on phytohormone activation or inactivation of aleurone layer metabolism and subsequent PCD. This is similar to H2O2 concentration changes observed recently by Ishibashi et al4. We have also successfully detected PCD induced by phytohormones in barley aleurone layer using a double-fluorescent probe-system also used by Fath et al5. Future challenges include integrating both these systems into a microfluidic device for plant tissue culture.