Real-time imaging of photosynthetic oxygen evolution from spinach using LSI-based biosensor.

The light-driven splitting of water to oxygen (O2) is catalyzed by a protein-bound tetra-manganese penta-oxygen calcium (Mn4O5Ca) cluster in Photosystem II. In the current study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to perform two-dimensional imaging of light-induced O2 evolution from spinach leaves. The employed Bio-LSI chip consists of 400 sensor electrodes with a pitch of 250 µm for fast electrochemical imaging. Spinach leaves were illuminated to varying intensities of white light (400-700 nm) which induced oxygen evolution and subsequent electrochemical images were collected using the Bio-LSI chip. Bio-LSI images clearly showed the dose-dependent effects of the light-induced oxygen release from spinach leaves which was then significantly suppressed in the presence of urea-type herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Our results clearly suggest that light-induced oxygen evolution can be monitored using the chip and suggesting that the Bio-LSI is a promising tool for real-time imaging. To the best of our knowledge, this report is the first to describe electrochemical imaging of light-induced O2 evolution using LSI-based amperometric sensors in plants.

Real-time monitoring of superoxide anion radical generation in response to wounding: electrochemical study.

BACKGROUND: The growth and development of plants is deleteriously affected by various biotic and abiotic stress factors. Wounding in plants is caused by exposure to environmental stress, mechanical stress, and via herbivory. Typically, oxidative burst in response to wounding is associated with the formation of reactive oxygen species, such as the superoxide anion radical (O2•-), hydrogen peroxide (H2O2) and singlet oxygen; however, few experimental studies have provided direct evidence of their detection in plants. Detection of O2•- formation in plant tissues have been performed using various techniques including electron paramagnetic resonance spin-trap spectroscopy, epinephrine-adrenochrome acceptor methods, staining with dyes such as tetrazolium dye and nitro blue tetrazolium (NBT); however, kinetic measurements have not been performed. In the current study, we provide evidence of O2•- generation and its kinetics in the leaves of spinach (Spinacia oleracea) subjected to wounding. METHODS: Real-time monitoring of O2•- generation was performed using catalytic amperometry. Changes in oxidation current for O2•- was monitored using polymeric iron-porphyrin-based modified carbon electrodes (φ = 1 mm) as working electrode with Ag/AgCl as the reference electrode. RESULT: The results obtained show continuous generation of O2•- for minutes after wounding, followed by a decline. The exogenous addition of superoxide dismutase, which is known to dismutate O2•- to H2O2, significantly suppressed the oxidation current. CONCLUSION: Catalytic amperometric measurements were performed using polymeric iron-porphyrin based modified carbon electrode. We claim it to be a useful tool and a direct method for real-time monitoring and precise detection of O2•- in biological samples, with the potential for wide application in plant research for specific and sensitive detection of O2•-.

Simultaneous Real-Time Monitoring of Oxygen Consumption and Hydrogen Peroxide Production in Cells Using Our Newly Developed Chip-Type Biosensor Device.

All living organisms bear its defense mechanism. Immune cells during invasion by foreign body undergoes phagocytosis during which monocyte and neutrophil produces reactive oxygen species (ROS). The ROS generated in animal cells are known to be involved in several diseases and ailments, when generated in excess. Therefore, if the ROS generated in cells can be measured and analyzed precisely, it can be employed in immune function evaluation and disease detection. The aim of the current study is to introduce our newly developed chip-type biosensor device with high specificity and sensitivity. It comprises of counter electrode and working electrodes I and II. The counter electrode is a platinum plate while the working electrodes I and II are platinum microelectrode and osmium-horseradish peroxidase modified gold electrode, respectively which acts as oxygen and hydrogen peroxide (H2O2) detection sensors. Simultaneous measurement of oxygen consumption and H2O2 generation were measured in animal cells under the effect of exogenous addition of differentiation inducer, phorbol 12-myristate 13-acetate. The results obtained showed considerable changes in reduction currents in the absence and presence of inducer. Our newly developed chip-type biosensor device is claimed to be a useful tool for real-time monitoring of the respiratory activity and precise detection of H2O2 in cells. It can thus be widely applied in biomedical research and in clinical trials being an advancement over other H2O2 detection techniques.