Research On The Preparation And Properties Of Immobilized AChE For Detection Of OPs In Seawater

Organophosphorus pesticides(OPs) are extensively used in agriculture due to their relatively low environmental persistence and easy hydrolysis. The widespread application of these pesticides had led an increase entrance into various water bodies, such as rivers, lakes and oceans, and caused harm to the environment. OPs pollutions in the coastal waters not only threaten the sustainable development of aquaculture industry, but also affect human health seriously through the human food chain. In recent years, on-site detection techniques for pesticides analysis have developed rapidly. Among these techniques, acetylcholinesterase(AChE) biosensors based on the enzyme inhibition are drawing more and more attention. AChE immobilization is a crucial step for fabricating biosensors, which decides the stability, sensitivity, selectivity and practicability of these biosensors. However, the recent immobilization materials are not perfect enough to be conducive to the use and promotion of enzyme sensors.Chitosan(CS), deacetylated product of chitin, is a linear polymer with high contents of reactive amino and hydroxyl functional groups. Chitosan has been widely used as an immobilization matrix owing to its extensive sources, low cost, chemical stability and good biocompatibility. Chitosan microspheres(CS-MS) not only maintain the good properties of chitosan, but also enhance the mechanical strength evidently. Thus, they would be more suitable for the preparation of immobilized enzyme used in the flow injection system.In this study, immobilized AChE was prepared by covalent cross-linking method using CS-MS as the carrier and glutaraldehyde(GA) as the cross-linking agent. The immobilization conditions were investigated. The optimal immobilization process was as follows:1g of CS-MS activated by 5ml of GA(0.5%,v/v) for 0.25h were mixed with PBS (pH7.0-7.5) containing AChE of 0.75U, then the mixture was agitated at 4℃for 24 hours. Under optimal conditions, the immobilized AChE exhibited an improved enzyme activity recovery of 81.94% compared with other parallel investigations. Then, the preparation conditions of CS-MS were also optimized by orthogonal experiments considering both the recovery of enzyme activity and physical properties of microspheres. It was found that the microspheres got their best performance when water and oil phase volume ratio was 1:4, stirring speed was 700r/min, dispersion time was 1h, GA amount was 0.5ml, cross-linking time was 0.5h.In addition, FT-IR analysis confirmed that AChE was immobilized into CS-MS successfully, achieved by the covalent link and hydrogen bond between microspheres and enzyme. Scanning electron microscopic examination revealed that CS-MS were basically spherical, and they could offer excellent support for enzyme immobilization due to their inner network structure. Besides, microspheres activated by GA had relatively smooth surface, which was conducive to immobilization.Moreover, immobilized AChE showed good adaptability in seawater, the optimum temperature for immobilized enzyme shifted toward higher value region, and suitable pH range did not show any shift. The resistance to salinity, thermal stability and storage stability were all enhanced after immobilization. The immobilized AChE lose only 17% of its initial enzyme activity after 60 days of storage in phosphate buffer solution at 4℃. The apparent values of Michaelis constant Km of immobilized AChE (46.18mmol/L) was found to be higher than that of solution enzyme (8.157mmol/L). The results indicated that the immobilized enzyme had lower affinity towards its substrate, which could be explained as the steric hindrance of the active site and diffusional limitation of substrate and products because of the partial changes of enzyme conformation during immobilization.In this study, the detection of OPs in seawater samples was studied preliminarily using immobilized AChE as biological recognition element. The results showed that the optimum inhibition time for methyl parathion, malathion and chlorpyrifos of immobilized AChE at 30℃was 30,20 and 20min respectively when the pesticide concentration was 100μg/L. Then the responses of immobilized enzyme to the pesticides were also investigated. The detection limits of methyl parathion, malathion and chlorpyrifos were 5.34,5.75 and 5.95μg/L respectively. Therefore, methyl parathion, which the immobilized enzyme was most sensitive to, was selected as a reference pesticide; and the toxic equivalency unit was calculated to be 5.63μg/L finally. Afterwards, toxic equivalency method was utilized to evaluated the equivalent concentrations of pesticides in seawater samples to reflect their comprehensive pollution levels. In summary, chitosan microspheres are promising carriers suitable for AChE immobilization because of higher enzyme activity retention, better mechanical property and abundant sources. The immobilized AChE showed good enzymatic properties in seawater, and could response to OPs with high speed and sensitivity. Toxic equivalency quantity (TEQ) derived from the reference pesticide through the toxic equivalency evaluation method would better reflect the comprehensive toxicity and integrated pollution level. Hence, toxic equivalency method show favourable application prospect in the rapid monitoring technology of OPs in seawater.