Construction Of Portable Biosensor And Its Application In Pesticide Residue Detection

With the advent of the digital technology revolution in agriculture,the development of efficient,sensitive and accurate pesticide residue detection methods has become an important technical support for the national implementation of the agricultural product quality and safety program and the realization of intelligent transformation of agriculture,and is of strategic importance for promoting the safety supervision of agricultural products,public safety monitoring and agricultural information construction in China.Traditional pesticide detection techniques are confined by the use of large precision instruments,making the detection processes expensive,complex,time-consuming,and susceptible to environmental interference,which are unsatisfactory to the requirements for real-time pesticide field detection(Point-of-Care Testing,POCT).Therefore,the design and establishment of rapid and sensitive,stable and low-cost portable pesticide detection device,have gradually arisen wide concern among academics and industries.One object of the present thesis is to provide a series of novel pesticide sensors with the merits of rapid response,high sensitivity and high specificity by taking the advantages of high specificity,biocompatibility,high stability and susceptibility to the modification with nanozyme of biosensors.Another object of the present thesis is to develop portable sensors for rapid on-site detection of pesticides by the employment of biosensors in combination with solid-phase carriers such as test paper,screen-printed electrodes and hydrogel kits.By the present thesis,not only new methodologies for the visualization of organophosphorus pesticides on-site detection are put forward,but also new ideas for the design of portable detection platforms and interdisciplinary applications are achieved.The main research contents of this thesis are as follows:1.A hydroxyl cobalt oxide nanozyme material with high stability and large surface area is designed and prepared,which is immobilized in paper-based sensors for the visual detection of organophosphorus pesticides and their poisoning markers.By making use of the mimetc peroxidase properties,the as prepared hydroxyl cobalt oxide nanozyme material is capable of serving as a catalyst for the reaction between chromogenic substrate 3,3?,5,5?-tetramethyl biphenyl and H₂O₂ which produces blue products.Wherein H₂O₂ can be hydrolyzed by acetylcholine under the catalysis of acetylcholinesterase and choline oxidase.Whereas organophosphorus pesticides can effectively inhibit the activity of acetylcholinesterase to prevent the production of H₂O₂,the system has no color response,according to the color change to build color sensor.The stability of the sensing system can be vastly enhanced by utilizing the highly tolerant enzyme-mimetic material cobalt hydroxide instead of the fragile natural enzyme.The detection limit for parathion methyl is 0.1 ng m L^-1.In addition,a detection system comprising a self-made test paper and a smartphone can capture the color signal change of the test paper,through color analysis software to achieve accurate quantitative analysis of pesticides.The said detection system has good selectivity and reproducibility,which makes a useful exploration for the application of nanozyme in the visualization and detection of pesticides.2.A high-performance enzyme cascade system pesticide sensor is constructed by making use of inorganic materials in combination with biological enzymes to jointly construct nanocomposites(organic-inorganic hybrid nanozyme).The enzyme cascading system is composed of acetylcholinesterase,choline oxidase and organic-inorganic hybrid nanozyme.The introduction of acetylcholine and chromogenic substrate(3,3?,5,5?-Tetramethylbenzidine)can make the enzyme cascading system produce color response,such that the construction of an enzyme-inhibited pesticide sensor is achieved.The sensor integrates screen-printed electrodes and colorimetric test strips,enabling direct qualitative analysis of pesticides based on color changes,followed by accurate quantitative detection through electrochemical technology.The thermal and chemical stability of natural enzymes is enhanced by enzyme fixation technology,further enhancing the storage stability of sensors.In addition,the use of multi-enzyme synergy shortens the transmission distance of the substrate,improves the efficiency of electrochemical reactions,so that the detection threshold to the level of fg m L^-1,and effectively enhances the sensitivity of the sensor.Therefore,the above discovery of the multi-enzyme cascading system indicates the potential application of the prepared sensor for trace pesticide residue detection.3.Oxalate,as a non-surfactant pesticide additive,plays an important role in the formulation of pesticide dosage forms and the maintenance of optimal potency of active ingredients.We use a biotemplate method to synthesize Mn O₂nanosheets with oxidase-like activity,which can trigger the color development reaction when the chromogenic substrate tetramethylbenzidine(TMB)is introduced.Meanwhile,oxalate induces the decomposition of Mn O₂nanosheets and causes the loss of oxidation ability,which leads to the color change of the system.By embedding Mn O₂nanosheets into the hydrogel for the construction of a stimulus-responsive kit,recording and quantification the optical images of the kit using a smartphone and the software Image J,a rapid detection of oxalate in the range of 0.8-800?mol L^-1 with a detection limit of 0.8?mol L^-1.Meanwhile,the kit can simultaneously screen 12 actual samples within 10 minutes.It can be seen that the portable gel kit designed in this paper provides the possibility for the on-site high-throughput detection of pesticide adjuvants.4.A concept of integrating a target-responsive hydrogel kit with a smartphone application was proposed.A quantitative analysis model between light signal and pesticide concentration was developed using nanozyme as a sensitive material to build a gel kit and a smartphone application for both data acquisition and analysis processing to achieve accurate real-time analysis and detection of organophosphorus pesticides.A rapid response colorimetric kit is constructed using a hydrogel as the solid phase carrier and Mn O₂nanozyme as the recognition element.Among them,the self-development smartphone application with dual functions of data acquisition and processing can convert the corresponding image information into grayscale values,and further calculate the linear relationship between the grayscale values and the concentration of paraoxon.Under the optimal test conditions,the detection limit of the sensing system for paraoxon is 0.5 ng m L^-1.The sensor applied a multi-enzyme cascade catalytic system to amplify the detection signal,which effectively improved the sensitivity.At the same time,the hydrogel's 3D network structure provides a relatively inert environment,which in turn improves the stability of the sensor.The portable kit-mobile phone sensing platformr provides a new means for the field detection of pesticides.