Immobilization Of Acetylcholinesterase On Mesoporous SBA-15 For Detection Of Organophosphorus And Carbamate Pesticide Insecticides

Insecticides are highly toxic substances,which are widely used in modern agriculture,and can cause adverse effect to human health.Thus,a fast,sensitive and cost-effective detection method of pesticide residues is essential to assure the quality of environmental and food safety.Based on the inhibitory effect of carbamate and organophosphorus pesticides on acetylcholinesterase?AChE?activity,AChE can be used to detect the two kinds of pesticide residues rapidly and sensitively.In this paper,the synthesized SBA-15/NH₂-SBA-15 was used as immobilized supports of AChE for the detection of pesticides residues.Firstly,highly ordered mesoporous SBA-15 was synthesized by using triblock copolymer PEO₂₀-PPO₇₀-PEO₂₀?P123?as a template and TEOS as a silica source in acidic conditions,then,being modified by amino group via APTES as functional reagent.The obtained sample was labeled as NH₂-SBA-15.The structure of the samples were characterized by XRD?FT-IR and other means.These results showed that the sample was synthesized successfully,and the pore size of SBA-15 is about 12.5 nm and for NH₂-SBA-15 is about 10.2nm.According to the result of TG-DSC,the amounts of amino group into the pores of SBA-15 were estimated to be 2.76 mmol/g.Secondly,using the synthesized SBA-15 and NH₂-SBA-15 as support materials,the immobilization conditions and its properties were optimized.The best condition for immobilization was obtained including 20 ^oC?0.1 M pH 8.0 PBS?1 mg/mL AChE?10 mg/mL supports?30 min?0.4%BSA and 1.5mM glutaraldehyde.NH₂-SBA-15?10.2 nm?exhibited the higher immobilization efficiency than SBA-15?6.1 nm?and SBA-15?12.5 nm?.And almost all of the AChE were entrapped into the carrier with no enzyme leakage.The immobilized AChE on NH₂-SBA-15 had the highest thermal stability,which was prepared by absorbing 0.4%BSA into the carrier after the immobilization of AChE,then cross-linking using glutaraldehyde.Furthermore,AChE-NH₂-SBA-15 retained 82.8%of its initial activity after storage at 25 ^oC for 60 days and 94.5%at 4 ^oC for 60 days,and can be reused for at least 6times with over 55.0%of initial activity.Finally,using carbaryl and trichlorfon as model compounds,the immobilized enzyme?AChE-NH₂-SBA-15?exhibited a wider linear range with 1.0×10^-3-10.0 ppm and 1.0×10^-5-1.0ppm respectively compared with free AChE.The limits of detection obtained for carbaryl and trichlorfon were 1.2×10^-3 ppm and 7.0×10^-5 ppm,lower than free AChE.The statistical analysis was studied by adding a certain amount of pesticides into immobilized enzyme.The obtained results indicated reasonably good recovery between 94-103%and relative standard deviation below 5%.