Construction And Preliminary Application Of Plant Small Molecule Electrochemical Sensor

Plants produce a series of small molecules with biological activities in the process of life,such as sugars,amino acids,plant hormones,etc.These small molecules play an important role in plant growth,development and environmental adaptation.Therefore,it is important to detect the content of these small molecules in plants for understanding their biological functions.Electrochemical sensors have the characteristics of high sensitivity,strong specificity,simple operation,easy portability and so on.In this thesis,two active small molecules,γ-aminobutyric acid（GABA）and glucose（Glucose）were taken as examples,their electrochemical sensors were constructed respectively,and the GABA content in cucumber seedling leaves and the glucose content in cucumber fruits were measured in vivo.The research results are as follows:1.The construction of GABA immunosensor and its in vivo detection of GABA content in cucumber seedlings under salt stress.Disposable screen-printed electrode（SPEs）were selected as the sensor substrate,and gold nanoparticles,ferrocene-graphene-carbon nanotube nanocomposite,dopamine,GABA antibody,and bovine serum albumin were modified to achieve specificity to GABA.Electropolymerization of gold nanoparticles can greatly improve the conductivity of the working electrode,ferrocene as a signal molecule can supply the current signal,and graphene and carbon nanotubes have high specific surface area and strong stability after mixing.Electropolymerized dopamine can form a polymer membrane on the surface of the working electrode,which can bind more antibodies and improve the detection range of the sensor.Finally,the addition of bovine serum albumin adds a layer of non-specific membrane and increases the anti-interference ability of the sensor.This enables the sensor to perform in vivo detection on plants.The results show that the sensor has a wide detection range with extremely low detection limit,and there is a good linear relationship between the peak current and the logarithm of GABA concentration from 10 a M to 100 m M.The regression equation for the standard curve isΔi_p（μA）=0.57 lg C_GABA（a M/L）+1.5 with a correlation coefficient of 0.995.The detection limit of this sensor is 1.995a M（S/N=3.0）.Compared with other GABA sensors,the immunosensor developed in this study is superior to other existing methods.Using the sensor,cucumber seedlings in the control group and salt stress treated group were tested in vivo.After testing,the GABA content in the cucumber leaves of the control group was 1.19*10^-10M-1.32*10^-9M,and the average content was 6.73*10^-10M;the GABA content in the cucumber leaves of thestress treated group was 2.88*10^-9M-2.82*10^-8M,the average content is 1.12*10^-8M.The average content of GABA in the stressed cucumber leaves was about 16.6 times that of the control group.The trend of change are consistent with the results of the amino acid analyzer,confirming that the developed immunosensor can reliably detect GABA in plants.Compared with traditional techniques,the information obtained by our proposed immunosensor can reflect the situation in plants,which provides a new idea for future in vivo detection of GABA in plants.2.Construction of glucose electrochemical sensor and in vivo detection of glucose content in cucumber fruit.Six-array electrodes were developed.The working electrode was composed of four platinum wires,which greatly improved the sensitivity of the sensor.CS has good biocompatibility and has been widely used for enzyme immobilization.The addition of glucose oxidase can specifically recognize glucose,thereby improving the recognition efficiency of the electrode.Finally,a naphthol solution（Nafion）was added dropwise to protect the working electrode.In this study,a one-step deposition method was adopted,in which glucose oxidase（GOx）and chitosan（CS）were mixed in a certain proportion and electrodeposited to the working electrode in one step.The results show a good linear relationship between the log value of glucose concentration and the log value of the corresponding current in the range of 1 m M to 100m M.The regression equation is:current（μA）=3.76Lg（concentration/m M）+0.83,and the correlation coefficient is 0.993.The lowest detection limit of the sensor was 32n M（S/N=3.0）.The sensor can therefore detect glucose levels in most plants.The electrochemical sensor was used to detect cucumber fruit in vivo,and the result showed that the glucose concentration of the fruit seven days after flowering was 27.783-28.408 m M,with an average value of 28.35 m M;the fruit glucose concentration of ten days after flowering was 34.119-35.683 m M,with an average value of 34.88 m M.The glucose concentration in cucumber increases with the increase of the fruit.The conclusion is consistent with the previous research,and the constructed sensor has a good application prospect in precision agriculture.