Wearable Electrochemical And Miniinvasive Method For In-vivo Sensing Of Hydrogen Peroxide Signalling Molecule In Tomato

H₂O₂ is a basic early warning signalling molecule for plants to respond to various stresses.Real-time monitoring the dynamic H₂O₂ in plants is of great significance for the study of signal transduction and signal integration mechanisms based on H₂O₂ molecule.Traditional detection methods for H₂O₂ in plants can be used for static detection.However,continuous H₂O₂ monitoring in plants is still a large challenge for plant biochemical information sensing.Therefore,it’s urgent to develop effective and accurate means for in-vivo continuous sensing of H₂O₂ in plants.The content of H₂O₂ in plants is low（10-100μM）.Firstly,the electrochemical sensing mechanism of H₂O₂molecule in plants was studied,and a composite sensing interface based graphene-platinum nanoparticles（Pt NPs）with high electrochemical activity was constructed.H₂O₂ in plants will lead to the production of ascorbic acid with antioxidant effect.Excessive ascorbic acid will increase the complexity of matrix and interfere with the sensing of H₂O₂.An antimatrix interference mechanism was studied and an anti-interference microelectrode sensing method based on Nafion-poly-o-phenylenediamine（Nafion-PPD）anti-interference interface was established.Breaking through the plant cuticle barrier through the sensing interface is the premise of in-vivo sensing.In order to prevent the interface damage during the breakthrough process,a hollow microneedle electrochemical sensor with interface protection was developed to protect the microelectrode sensing interface.And the feasibility of using microneedle electrochemical sensor to sense H₂O₂ signal molecule in plants was studied.The relative displacement between sensor and plant tissue in the process of in-vivo sensing results in signal distortion.Therefore,a self-adhesive and wearable electrochemical microneedle sensor was developed,and a wearable electrochemical and miniinvasive method for in-vivo continuous sensing of H₂O₂ signal molecule in tomato was established.Main research contents and results are as the following:（1）Electrochemical sensing mechanism of H₂O₂ and construction of graphene-platinum nanoparticle composite sensing interfaceAiming at the low content of H₂O₂ in plants,the electrochemical sensing mechanism of H₂O₂was studied and a graphene-platinum nanoparticles（Pt NPs）composite sensing interface was constructed by laser induced graphene（LIG）technology.The influence of laser parameters on the morphology of LIG was studied,and the electrochemical deposition conditions of Pt NPs were optimized.The catalytic performance of the composite sensing interface was characterized.Results show that Pt NPs can be effectively deposited on the surface of laser induced graphene sheet.The nanocomposite sensing interface exhibites good linear relationship with H₂O₂ in the range of 2-200μM,and the limit of detection（LOD）is 0.35μM.The influence of matrix effect on the sensing performance of nanocomposite sensing interface was explored,through the in-vitro simulation in tomato bleeding sap,Under the simulation condition in bleeding sap,the sensing interface can detect at least 5μM H₂O₂.（2）Antimatrix interference mechanism and construction of Nafion-PPD anti-interference interfaceFor the requirements of antimatrix interference interface and size of implanted device.The antimatrix interference mechanism of interface was explored.The graphene-Pt NPs composite sensing interface was constructed on the surface of platinum microcolumn electrode,and the modification conditions of the composite sensing interface was optimized.Double-layer based anti-interference films were constructed at the composite sensing interface.The influence of the modification order of the double anti-interference films on the anti-interference performance was explored,and the double-layer films based anti-interference strategy and method were established.Results show that the nanocomposite sensing interface-based microelectrode exhibites good linear relationship with H₂O₂in the range of 0.5-2000μM,with the LOD of 0.33μM and response time less than 1.0 s.The nanocomposite sensing interface-based microelectrode processes higher sensitivity,stronger anti-interference performance and smaller interface size,which lays a research foundation for the construction of miniinvasive sensor devices and the in-vivo continous sensing of H₂O₂ in tomato.（3）Construction and analytical performance of hollow microneedle electrochemical sensor with interface protectionAiming at the problem of sensor interface damage during miniinvasive breakthrough of plant cuticle barrier,a hollow microneedle electrochemical sensor with interface protection was developed.The hollow microneedle was prepared by 3D printing technology.Meanwhile,the reference electrode and counter electrode at the micron level were prepared.The platinum microelectrode modified with the graphene nanocomposite sensing interface was used as the working electrode,and the microelectrode system was integrated into the central holes of the microneedle to form the hollow microneedle based electrochemical sensor.The influence of matrix effect on the sensing performance was investigated through in-vitro simulation of bleeding fluid.The microneedle based electrochemical sensor was installed on tomato plants under osmotic stress,which verified the feasibility of the microneedle based electrochemical sensor for sensing H₂O₂ signal molecule in plants.Results show that the sensing interface can detect at least 10μM H₂O₂ in bleeding sap.The hollow microneedle opens up a physical channel for microelectrodes to break through the cuticle barrier and interact with sap of vascular bundle,which provides technical support for establishing in-vivo continous sensing method for H₂O₂in plants.（4）Construction of self-adhesive wearable electrochemical microneedle sensor and in-vivo continuous sensing of H₂O₂Aiming at the problem of signal distortion caused by relative displacement between sensor and plant tissue in the process of in-vivo sensing.A self-adhesive and wearable electrochemical microneedle sensor was developed,and a wearable electrochemical and miniinvasive method for in-vivo continuous sensing of H₂O₂ in tomato was established.The microelectrodes were integrated into the dissolving polymer microneedle to prepare sensing patch.After moisture absorption,microneedle base can be softened and coherently adhere to plant leaves,stems and other tissues.The mechanical strength of the needle,the tissue adhesion,and the anti-disturbance performance of the device were characterized.Results show that the mechanical strength of the microneedle is sufficient to ensure that the needle penetrates the plant tissue.The needle dissolves in the plant tissue,thus exposing the sensing layer for monitoring H₂O₂.The electrochemical microneedle sensing patch has the characteristics of sensing interface protection,strong tissue adhesion,and interface conformability.It can monitor the H₂O₂ in plants under stress conditions.This study provides a new way for mininvasive and in-vivo continous sensing of H₂O₂ in plant.