E-mail address:(Y.Xu).

Organophosphorus(OPs)and carbamates as pesticides are w idely used in modern agriculture[1].But they are highly toxic substances,w hich can cause adverse effects to human health through the transmission of food chain,resulting in malignant diseases[2].Acetylcholinesterase(ACh E)is mainly found in cholinergic neurons and can catalyze hydrolysis of acetylcholine into choline and acetate the enzyme is inhibited by many organophosphorus and carbamate pesticides,such as trichlorphon,carbaryl and carbofuran[3].Thus,fast,sensitive and cost-effective detection of pesticide residues is essential to assure the quality of environmental and food safety.

The free ACh Ehas some disadvantages w ith high-cost expenses,poor stability,can only be stored at-20°C and be used only of ACh E on carrier is an effective method to solve the existence materials have been developed for enzyme immobilization,including magnetic particles[4–6],metallic/metallic oxide nanoparticles[7–12],carbon nanotubes and hybrid carbon nanotubes nanocomposites[13–15],inorganic membranes[16,17]and others[18–20].Although they have high sensitivity and precision,most w ere made into sensors and stored at 4°C.The products of pesticide residue detection,w hich w ere sold in domestic markets at present,had the defects of low sensitivity and needed to store at low the immobilized ACh E can be very stable at room temperature,the application area for pesticide residue detection reagent w ill be extended w ith a low er cost of transportation and ,it is very essential to develop a stable detection reagent w ith high precision and sensitivity,w hich can be stored at room temperature for a long time.

Mesoporous silica materials w ith its inherent characteristics of high surface area pore volume,tunable pore size accommodating dimensions of enzymes,and mechanical stability,has been regarded as a proper vector for immobilizing[21–25].The enzyme,ACh E,had been already immobilized on calcined mesoporoussilica m aterials[17,26,27],and almost all of them w ere prepared into a sensor,w hich was required a lot of energy during the calcinations process,and the preparation of immobilized enzyme w ere quite complicated even w hich could only be stored at low re fluxing is another way to remove the template of the as-synthesized material,and moderate amount of the template is bene fit for immobilizing[28].Compared to other silica materials commonly used,functionalized mesoporous silica SBA-15 molecular sieve has the advantage of preventing the enzyme leaching from the carrier and increasing the activity of the enzyme,w hich is arising from the strong electrostatic interaction betw een the enzyme and the surface of the supporter[26,28–32].To the best of our knowledge,no report w as found for the immobilization of ACh E on functionalized SBA-15.

In this article,the SBA-15 and NH2-SBA-15 mesoporous molecular sieve w as explored as immobilized supports of ACh E for the detection of pesticides order to obtain a stable,facile immobilized enzyme that can be stored at room temperature,the optimum immobilization method and condition of ACh E w ere properties of immobilized enzyme w ere also explored application of immobilized ACh E in the detection of pesticides w as also investigated and compared w ith free ACh E.

Highly ordered mesoporous SBA-15 w as synthesized according to the literatures[27,33]by hydrothermal synthesis using triblock copolymer P123 as a template and TEOS as a silica source in acidic conditions.

The surface of SBA-15 w as modi fied by amino group via re fluxing of APTES and TEOS(1:10,molar ratios)in dry toluene at 110°C for 24 h w ith stirring under w as w ashed w ith dichloromethane and dried in obtained sample w as labeled as NH2-SBA-15.

The immobilization of ACh E on SBA-15 or NH2-SBA-15 w as performed in tw o methods.

Covalent coupling method:In each experiment,10 mg/m L NH2-SBA-15 reacted w ith 1.5 mmol/L of glutaraldehyde for 2 h at 30°C in PBS(0.1 mol/L,p H 8.0),and the suspension was mixed w ith 1 mg/m L of ACh E solution for another 30 min.The immobilized enzyme w as separated by centrifugation,w ashed w ith PBS and suspended w ith 0.4%BSA for another 30 ,the immobilized enzyme was obtained by centrifugation,w ashing and rinsing,and labeled as NCC for NH2-SBA-15 immobilization of ACh E by covalent cross-linking method.

Adsorption-crosslinking method: In each experiment,10 mg/m L NH2-SBA-15/SBA-15 w as mixed w ith 1 mg/m L of ACh E solution in 0.1 mol/L phosphate buffer(PBS)at p H 8.0 for 30 min.Then,the immobilized enzyme w as separated,w ashed and suspended w ith 0.4%BSA for another 30 centrifugation and w ashing,glutaraldehyde( final concentration,1.5 mmol/L)w as mixed w ith the immobilized enzyme for 2 h to achieve covalent ,the immobilized enzyme w as rinsed three times w ith PBS and labeled as NAC/SAC respectively for NH2-SBA-15 immobilized ACh E by adsorption-crosslinking method and SBA-15 immobilized ACh E by the same adsorption-crosslinking method.