| Organophosphate and carbamate pesticide is widely used in agricultural production due to its advantages of high efficacy, low toxicity and easy degradation. But too much pesticide residues will not only cause serious food safety problems, affect people's physical health, but also cause too much trade barriers. At present, chromatography detections of pesticide residue such as gas chromatography, high performance liquid, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry have the advantages of sensitive and accurate, but these methods need high cost of equipment, long testing period, and operating personnel. Therefore to development of rapid detection technology, block pesticide residues on food outside of the table, and to ensure food safety of our people have the very vital significance.With immobilized acetylcholinesterase (AChE) by chitosan microsphere working as the recognition element, a novel flow analysis chemiluminescence (CL) sensor for the detection of organophosphorous and carbamate pesticides was developed and optimized. The details of the contents and results of the thesis are as follows:1 Chitosan microspheres (CS-MS) was used as the carrier for immobilization of AChE, with glutaraldehyde (GA) as the cross-linking agent. The preparation conditions of CS-MS was optimized, the results found that when the water and oil volume ratio was 1:4, the rotational speed was 1100 r/min, the chitosan concentration was 5%, and the dispersant was 1.5 mL, the preparation of microspheres was better in performance. Before immobilizing, the AChE was activated with 2 mL 0.4% GA solution of CS-MS for 15 min, and the optimal fixed method was:add 0.6 U free enzyme into 0.500g CS-MS activatived by GA, then shocked slowly in PBS (0.01 mol/L, pH=8), and kept shocking for 20 h under 4?. It was also found that the immobilized enzyme has stronger adaptability of thermal stability and the pH than the free enzyme system. But, the immobilized enzyme had a lower substrate affinity. Two months later, the immobilized enzyme activity dropped 18.19%. Immobilized enzyme had good reproducibility, and the average deviation was less than 5.74%.2 The organic polymer polymethyl methacrylate (PMMA) was used as the material for fabricating microfluidic chip, which consisted of a base plate with the size(100 mm× 700 mm×4 mm) and a cover plate with the size (100 mmx70 mmx2 mm). We adopted the milling machining to produce the micro structure. The base chip has a spiral channel and four holes of diameter 1.8 mm. The channel, with the width of 0.8 mm, the depth of 1 mm and the space between channels of 0.9 mm, was produced by etching. Four holes were used as the inlet (center) and outlet (side) respectively. The bonding of chip was accomplished by solvent bonding method at room temperature. The procedures were as follows:the base plate and cover plate were cleaned with ultrasound in water for 10 min. After the chip dried, a few drops of dichloroethane/anhydrous alcohol (1:1, V/V) mixture was added on the contact surface of the cover plate and coated uniformly. Then, the base plate and cover plate were aligned, and pressed at room temperature for 5 minutes. After bonding, black ink was injected into the micro tube and no leakage is found, which indicates that the chip's contact was compact after the bonding. Then the chip was dropped down freely from a height of 1.5 m onto the concrete floor and the chip was intact, and the strength of the chip was over 100 N/cm2, which indicates that the impact strength of the bonding meets the requirement.3 In this study, the flow injection-chemiluminescence systems, including a reactor with immobilized AChE on CS-MS and a miniaturized flow cell detector, was proposed for the detection of organophosphate and carbamate pesticide. A set of experiments were conducted to evaluate the detection performance of the system with different assay parameters. After optimizing the experimental conditions, such as luminol concentration, ferricyanide concentration, flow rate, substrate concentration, substrate injected time, enzyme-pesticide contact time, it was found that the developed FIA-CL system worked well for quantified assays. As a result, the optimum condition was obtained. Under the optimum condition, dichlorvos as a kind of organophosphate pesticide and carbamate pesticide were detected. The inhibition rate of dichlorvos was proportional to its concentrations in the range of 0.08?10.00 ?g/mL. Regression equation was Y=22.837X+33.945 and correlation coefficient was 0.9943. The detection limit was 0.053 ?g/mL; the inhibition rate of carbofuran was proportional to its concentrations in the range of 0.08?15.00 ?g/mL. Regression equation was Y=20.801X+30.726 and correlation coefficient was 0.9937. The detection limit was 0.058 ?g/mL, and the inhibited enzyme was reactivated by 1 mmol/L pyridine-2-aldoxime methiodide (2-PAM).To further demonstrate the applicability of the AChE-based CL biosensor, the recovery and reproducibility test was studied by adding pesticides into vegetable samples. The results showed that the biosensor has good recovery (>80.9%) and reproducibility (RSD<8%, n=6). The enzymatic reactor can be used more than 18 times. It's good for the commercialization of the sensor. In addition, the principle of biosensor was discussed to a certain extent. |
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