Construction Of Enzyme-based Portable Biosensor And Its Application In Pesticide Detection

In agricultural production,pesticide can prevent,control and eliminate diseases,insects and weeds to guarantee stable and increased crop yield.However,the long-term,large-scale use and improper abuse of pesticides will induce environmental pollution and lead to ecological security risks.In particular,long-term exposure to pesticides and enrichment of the food chain can cause dysfunction of central nervous regulation,or even death by poisoning,seriously threatening human health.Therefore,detection of pesticide exposure markers,including pesticide molecules and organism biomarkers,is of great significance for food/environment monitoring and pesticide poisoning early warning.At present,there are a variety of detection methods for pesticide exposure markers,such as chromatography,immunoassay and enzyme inhibition method.Although these methods can achieve accurate detection of targets,they require sophisticated and expensive instruments,professional operators and complex operation steps,etc.,which limit their application in field detection.Focus on the above problems,the rapid development of nano science and technology to build high-performance biosensor provides an opportunity to biological sensors based on nanomaterials are compared with the large analysis instrument with high sensitivity,simple operation,quick response,and low price advantage,especially can be used to assemble a portable sensor in order to realize the scene real-time detection.Therefore,it is widely concerned in the field of sensing and detection and has become the research frontier and hotspot in this field.One of the key problems faced by portable biosensors is how to make full use of the characteristics and advantages of different sensitive materials to improve the sensitivity and stability of sensors and further realize field application.This thesis is devoted to the research of a new method for the field detection of pesticide exposure markers,and the construction of portable biosensor by the fusion of nanomaterials and biological enzymes.Specific research contents are as follows:1.A portable biosensor for the field detection of acetylcholine（body biomarker）was constructed using stimulus-responsive hydrogels.Using Cu₃（PO₄）₂as an inorganic carrier framework and bovine serum albin as an organic unit,organic-inorganic hybrid nano-flowers with peroxidase-like activity were prepared by biomineralization,which could catalyze the oxidation of-3,3’,5,5’-tetmethylbenzidine（TMB）in the presence of hydrogen peroxide to give the blue oxidation state TMB（Ox TMB）.The organic-inorganic hybrid nano-flowers and TMB were simultaneously encapsulated in agarose hydrogel,and a portable"reaction tube"was developed for visual detection of acetylcholine.Combined with smart phones and image processing software,the accurate detection limit of acetylcholine in human serum was 0.35μmol L^-1.It provides a new platform for on-site screening of pesticide poisoning.2.By embedding copper nanoparticles into the three-dimensional network of agarose hydrogel,the urease stimulation response gel kit was constructed,and the portable detection of dimethoate pesticide was realized.As an inhibitor of urease,dimethoate prevented ammonia production and reduced in situ etching of copper nanoparticles,resulting in fluorescence response of the gel kit.The fluorescence color information was collected by smart phone,and the image information was converted into digital signal by Image J software.The detection limit of dimethoate was 1.0μg L^-1and high sensitivity was realized.By fusion agarose hydrogel kit with the color of the smart phone recognition system,not only improves the detection sensitivity and stability,but also simplifies the operation process and shorten the analysis time of the sample（working time only 55 minutes）,this method can well meet the requirements of daily test,for the site provides a new method for monitoring food safety and human health.