Enzyme Labeled Pesticide Used In Thermal Enzymatic Biosensor For Pesticide Residue Detection

The food safety incidents caused by pesticide residues frequently occur in food or agricultural products, and bring great loss of personnel and property, which turn to the social hot issues deeply concerned by all levels of government and the people. There are some limitations in the electrochemical or spectrophotometric method commonly used in detecting pesticide residues. They are susceptible to electrochemically active components or optical materials, resulting in false positive.The Calorimetric sensor detection method has the advantages of simple operation and strong versatility, which is able to overcome the above defects, but it is difficult to apply in measuring pesticide residues because of the heat release of the enzyme-substrate reaction is very small in the enzyme inhibition and poor in sensitivity. We need to find an effective method to amplify the related reaction heat release to solve the above problems and improve the measurement accuracy. Using the enzyme to label the pesticide will increase heat release of whole detection system in enzyme inhibition method by the heat reaction of marker enzymes.It may be one of the way to improve calorimetric accuracy.The accurate determination methods of enzyme reaction enthalpy using micro-calorimeter had been studied in this thesis. And we use this method to screen the proper enzyme and its reaction system. The methamidophos pesticide labeled by catalase was prepared using carbodiimide method. We took inhibition rate of enzyme labeled pesticides on immobilized chicken liver esterase and marked enzymes activity per inhibiton rate as the standards to optimize the preparation process.Firstly, taking the catalase catalyzed hydrogen peroxide reaction as an example, the synchronized calorimetric and spectrophotometric method for measuring enzymatic reaction enthalpy had been studied systematically. The difference between initial velocity calculation method and steady-state calculation method in calculating enzymatic reaction enthalpy was described. Compared with the theoretical values and reported values, the catalase catalyzed hydrogen peroxide reaction’s enthalpy using the steady-state calculation method was 100.98KJ/mol, the standard deviation was 1.81 KJ/mol, showed more accurate and demonstrated better reproducibility. The two methods used a same enzymatic reaction system to simultaneously determine the process of reaction from the perspective of optical and thermal characteristics of the enzymatic reaction, reducing the experimental errors.Secondly, the method was used to determine different enzymatic reactions’ enthalpies, such as catalase catalyzed sodium perborate, ?-lactamase catalyzed penicillin G sodium reaction, glucose oxidase catalyzed glucose reaction. We found that the method to measure glucose oxidase catalyzed glucose reaction’s enthalpy was inproperty because of non-specific heat which caused by mechanical friction, solution mix and other reasons. The result of the two front was 108.98KJ/mol,90.67KJ/mol, the standard deviation was 5.87 KJ/mol,4.63 KJ/mol, respectively, except for glucose oxidase catalyzed glucose reaction. According to the screening criteria of marker enzymes and the principle of reaction heat larger, catalase was selected as the marker enzyme and sodium perborate as the substrate.At last, it was successfully prepared catalase labeled methamidophos pesticides using carbodiimide as crosslinking agent. The preparing methods of enzyme labeled pesticides were optimized according to inhibition rate of enzyme labeled pesticides on immobilized chicken liver esterase and marked enzymes activity per inhibiton rate. The optimal reaction conditions of catalase labeled methamidophos were identified: The molar ratio of catalase and methamidophos 1:10; The conjugation reaction of the enzymes with crosslinking agents at 30℃; The 0.05 M MES buffer(pH6)which was used in the reaction of enzyme with crosslinking agent; The concentration ratio of crosslinking agent(EDC and NHS) 5 mol/L: 5 mol/L; The p H value was not adjusted in the reactions; The reaction temperature of enzyme derivatives and methamidophos at 20℃.