Real-Time Monitroing Of Plant Signaling Molecules By Microelectrode Electrochemistry

Plant hormones and reactive oxygen species (ROS) play crucial roles in mediating the whole stage of plant development, ranging from seed germination, stem elongation and flowering to cone production and apoptosis. Because of the tiny amount and high spatial and temporal dynamics of plant hormones in quantities and composition, it is quite indispensable to develop in-situ and real-time methods to detect plant hormones on the level of plant organs, micro-organisms and even single cells to provide powerful tools to unravel mechanisms of their function, metabolism, signal transfer and regulation. Owing to the properties of rapid pesponse, high sensitivity and resolution, ultramicroelectrode has made important progress in real-time detection of single mammalian cells. However, it hasn't been successfully used in measuring hormones of plant cell for lack of high sensitive electrochemical sensor. Recently, nanomaterials have been widely used for improve the sensitivity, selectivity and stability of electrochemical sensors, due to its large specific surface area, high surface free energy and fast rate of electron transfer. To meet the urgent requirement of in-situ and real-time detection of plant hormones and the unique advantages nanomaterials displayed in electrochemical sensors, this work fabricated modified ultramicroelectrodes by using metalnanoparticle, nanowire, graphene and their composite materials to construct high-performance sensors to detect plant hormone in real-time from single plant cells, which was further used to initially study of regulatory mechanism of oxidative burst and the vesicle fusion of auxin exocytosis. The major works are as following:1. With Nafion as a template, uniform Pt nanoparticles were electrochemically deposited on the surface of carbon fiber microdisk electrode (CFMDE), forming the NPt/Nafion/CFMDE electrode. On the other hand, an agarose microarrayfluidic chip, which was used for efficient immobilization of plant cell suspension, was fabricated based on polydimethylsiloxane (PDMS) by soft lithography. NPt/Nafion/CFMDE was used to monitor detect real time oxidative burst of a single Arabidopsis thaliana protoplast in real-time, and results confirmed that the major component of ROS was hydrogen peroxide. Then, protoplast was treated with thin layer liquid culture to recover cell wall. The results indicated that intact plant cells released much less H2O2molecules with faster kinetics to the extracellular environment compared with single protoplasts, while the ROS level could be recovered when the activity of cell wall peroxidase (POD) was inhibited. The results provide evidences that cell wall participates in regulating transient oxidative burst transient by a mechanism involving cell wall POD machinery at single cell level. We think the results presented here would facilitate better understanding on ROS burst and their regulatory mechanisms.2. Using titanium alloy (Ti6A14V) as basal material,TiC@C NWA microelectrode was prepared and the surface was then coated with Pt particles of3-6nm in diameter by chemical reduction, forming the TiC@C/Pt-QANFAS electrode. The resulted electrode was further modified by AZ5214insulating cement and then only the tip of electrode was exposed to reduce effective surface and enhance the ratio of signal to noise. This electrode showed high sensitivity to plant hormone auxin, and the detection limit was1nmol/L. The novel electrode was further used to in real-time monitor the release and kinetics of auxin efflux from single rapeseed protoplast for the first time. It has demonstrated that auxin is released to extracellular environment via exocytosis, and there are full collapse fusion (FCF) mode and kiss and run (K&R) mode. This sensitive and rapid-response sensor can be applied to study polar auxin transport.3. Using CFMDE as basal electrode, NAu/CFMDE was prepared by depositing an uniform layer of nano Au. It was shown that this electrode showed good linear relationship between2.0×10-7and7.5×10mol/L when analyzed salicylic acid, and detection limit is1.5×10-7mol/L. Selectivity of this electrode was good and this measurement was not interfered by ascorbic acid. Application of this electrode for measurement of salicylic acid in plant extract gave satisfactory results. This technology is likely to be applied to study mechanism of system acquired resistance induced by salicylic acid.4. Using carbon fiber cylinder electrode as basal electrode, composite nanomaterial of graphene and nano Pt particle was embedded on surface via cyclic voltammetry method, and NPt/rGO/CFMCE electrode was prepared. Compared to basal electrode, sensitivity of NPt/rGP/CFMCE improved about7fold, showing good linear relationship in range of1.8×10-9-1.5×10-8mol/L, and detection limit is1.5×10-9mol/L. It has been applied to determinate concentration of abscisic acid in Magnolia denudate leaf.