Metal-Organic Framework Based Fluorescent Sensing Methods For Signal Molecules Of Plant Disease And Pest Stress

Plant disease and pest seriously affect the growth and development of crops,resulting in serious reduction of crop yield.Sensing the information of the disease and pest stress in the early stage and taking approaches to resist the stress is an effective method to reduce crop yield loss.The plants have a variety of immune systems to defend the attack of pathogens and pests.These immune systems work by the recognition and transmission of signal molecules.During the immune process,the concentration of the signal molecules will be changged.Hence,detecting the fluctuations of signal molecules in plant is a valid approach to obtain the early stress information.Until now,two kinds of in-situ detection methods including electron paramagnetic resonance spectroscopy(EPR)and fluorescence sensor have been applied to detect the signal molecules in plant.However some limitations have been existed in these methods to restrict their applications.For example,the EPR probes suffered from low specificity.The fluorescence sensor had the problem of the autofluorescence interference of plant.The genetically encoded fluorescent protein probes in fluorescence sensors is only suitable for model plants.In view of this,it is urgency to develop an sensor for detecting the variation of crop(non-model plants)signal molecules of pathogen and pest stress in vivo.This sensor should have excellent detection performance and avoid the interferences from plant autofluorescence in vivo.Plant nanobionics sensor refer to infiltrating nanomaterials with special functions into plants to react with signal molecules and generating the detectable signals.Plant nanobionics sensor is a general detection technology that can real-time monitor the spatiotemporal information of signal molecules in model and non-model plants.In this thesis,using plant pathology as the research foundation,plant nanobionic and fluorescent technique as the research tools,starting from the interaction mechanism between nanomaterials and signal molecules,two sensing methods based on metal-organic framework(MOF)composites have been developed.High-sensitive and in vivo real-time sensing of signal molecules induced by plant disease and pest stress were gradually realized.After figuring out the sensing mechanism and optimizing the parameters,the optimal probes were infiltrated into the plant to establish a near-infrared fluorescent plant nanobionic sensor.The constructed plant nanobionic sensor can monitor the spatiotemporal information of the signal molecules induced by plant disease and pest stress.The sensor can also study the signal pathways of these signal molecules.The plant nanobionic sensors give a new idea for sensing other signal molecules in plants.The sensor also provides new ways and means for the implementation of intelligent agriculture.The main research contents and findings of this thesis are as follows:(1)In order to realize catalytic fluorescence sensing of hydrogen peroxide as a signal molecule under stresses of plant disease and pests with high sensitivity,the catalysts with high activity have been designed and synthesized.Due to the advantages of the synergistic catalytic effect between two nanomaterials,noble metal/metal-organic framework nanocomposites with was prepared.Two approaches including surfactant-assisted synthesis and shear exfoliating were applied to prepare various two-dimensional MOF(2D MOF)nanosheets.The 2D MOF nanosheets with larger specific surface area,less surface defects and higher yield were selected as the substrates to grow a variety of noble metal nanosheets with tunable morphologies through a template method.By structural design and parameter optimization,the nanocomposites provide the basis for the development of a catalytic fluorescence sensing methods for detecting hydrogen peroxide with high sensivity.(2)The Pd/Cu-TCPP(Fe)nanocomposites with highest catalytic activity was selected,which were further used to construct a highly sensitive detecting method for sensing hydrogen peroxide.The Pd/Cu-TCPP(Fe)nanocomposites displayed a higher catalytic activity compare to its corresponding individuals,proving its synergistic catalytic effect.The steady-state kinetic analysis demonstrated that the Pd/Cu-TCPP(Fe)nanocomposites possess peroxidase-like activity.The catalytic activity of Pd/Cu-TCPP(Fe)nanocomposites was used to catalyze the substrate Amplex Red to generate fluorescence in the presence of hydrogen peroxide.The fluorescence intensity is proportional to the concentrations of hydrogen peroxide.Based on this detection mechanism,a highly sensitive sensor for detecting hydrogen peroxide was constructed,and the detection limit of the sensor is 0.369 μM.(3)The fluorescence of the probes in the previous chapter is in the visible light window,which seriously suffered from the interference of plant autofluorescence.To solve this problem,a near-infrared fluorescence detecting method based on metal-organic framework nanocomposites is designed and constructed.The commerial fluorescent dye IR-1061 of the near-infrared region II was utilized to prepare the water soluable IR-1061 micelles through rotary evaporation.The IR-1061 micelles were further embedded in MOF(ZIF-90)through an one-pot method.The layer of ZIF-90 significantly enhanced the fluorescence intensity of IR-1061 micellces and could recognize ATP with high specificity.Hence,the prepared MOF nanocomposites were used to construct a near-infrared fluorescence sensor,which pocess highly sensitive(detection limit: 0.27 m M)and highly specific detection of ATP.Fluorescence in the near-infrared window can effectively avoid the interference from plant autofluorescence,which establish the foundation for the subsequently real-time sensing of extracellular ATP as a signal molecule under stresses of plant disease and pest.(4)The prepared MOF nanocomposites were infiltrated into plants to construct a plant nanobionic sensor.The nanobionic sensor realized the spatiotemporal information monitoring of the extracellular ATP.A near-infrared fluorescence imaging system and a near-infrared fluorescence microscopic imaging system were built to sense the fluctuation of near-infrared fluorescence signal and confirm the distribution of nanocomposites in plants,respectively.The MOF nanocomposites were infiltrated into the spinach leaf through needleless injection.The distribution of infiltrated MOF nanocomposites focus on intercellular substance in spinach leaf.By exogenously applying ATP,the nanobionic sensor have a detection range of 0-5 m M in spinach and scindapsus aureus,and 0-2 m M in pepper.Based on the excellent sensig performance,the constructed nanobionic sensor realizes real-time monitoring of extracellular ATP under the stresses of plant disease and pest stress.