Immobilization of acetylcholinesterase on f(3)

Table 2 Detection of pesticides content in standard solution and green vegetables by ACh Samples Added(ppm)Found(ppm)Recovery(%)RSD(%)Carbaryl Standard 2.0×10-31.87×10-393.8 4.82 Vegetables 2.0×10-31.94×10-397.1 3.88 Trichlorfon Standard 1.0×10-41.03× 3.91 Vegetables 1.0×10-41.09× 4.76

The reusability and storage stability of immobilized enzyme are tw o of the most vital features in practical application,primarily due to its commercialization demands that enzymatic process should economically reduce (Supporting information)show ed the recycling results of three kinds of immobilized ACh E.For NCC and SAC,44%and 45%residue activity w ere found after being recycled for 6 times, ever,it gave the 55%of initial activity for NAC.This result w as comparable to that of ACh E immobilized on porous silicon[40].

The storage stability of the lyophilized immobilized enzyme,w hich w as stored at 4°C and 25°C w as show n in Fig.1,NAC retained 94.0%and 82.8%of its initial activity after storage at 4°C and 25°C for 60 days.The results w ere signi ficantly improved compared w ith the previous reports[16,41,42].It indicates that this immobilization method can retard the aggregation or denaturation processes of is a strong possibility that ACh E in the pore of NH2-SBA-15 is very good to retain the structure and ,they cannot be altered as freely as they are in solution because the pore sizes are close to the size of ACh E results illustrated this proposed immobilized enzyme could be stored w ith longer time at room temperature,w hich not only could conserve energy but also be easy for reusing.

The application of NAC(ACh E-NH2-SBA-15)w as examined by different is w ell know n that organophosphorus and carbamates pesticides inhibit ACh E ,immobilized ACh E can be used for detecting various pesticides and its inhibition is directly related to the amount of pesticides[43]. Figs.S9 and S10(Supporting information)show ed the effect of concentrations of inhibitors on the activity of ACh carbaryl curve gave a linear relation for the range 1.0×10-3ppm to 10.0 ,the linear relation of trichlorfon is 1.0×10-5ppm to 1.0 ppm.The limit of detection(LOD)obtained for carbaryl and trichlorfon w ere 7.6×10-4ppm and 2.4×10-6ppm, for free ACh E,the linear range of carbaryl is 5.0×10-3ppm to 5.0 ppm,and trichlorfon is 1.0×10-3ppm to 1.0 ppm,furthermore the LOD is 2.0×10-3ppm for carbaryl and 9.0×10-4ppm for trichlorfon (Table 1).It can be seen from results that the immobilized ACh E has higher sensitivity,w ider linear range and low er LOD than free ACh E.

The detection of pesticides residues on standard pesticide solution by ACh E-NH2-SBA-15 w as conducted 7 times for the same sample,and 3 times on real vegetable sample for the calculation of relative standard deviation(RSD), can be seen from Table 2,the current method show ed good recovery(93.8%-109.3%)and low RSD(＜5%).Therefore,this proposed immobilized ACh E can be regarded as a promising reagent for pesticide residues detection of real samples.

In summary,a simple,effective adsorption-crosslinking method has been developed to immobilize ACh E on mesoporous NH2-SBA-15 molecular NH2-SBA-15 show ed 95%immobilization ef ficiency and the speci fic activity of immobilized ACh E(NAC)reached 130%relative to free ACh E.The immobilized ACh E also show ed good reusability and high storage ,using carbaryl and trichlorfon as model compounds,the immobilized enzyme exhibited a w ider linear range,low er limit of detection than free ACh E for the detection of the accuracy and precision is also good for determination both of the standard solutions and real vegetable a w ord,the ACh E immobilized on NH2-SBA-15 is a promising reagent tow ards pesticides residue detection for its fast,sensitive,stable and lowcost characters.

Acknow ledgments

This w ork w as supported by the National Natural Science Foundation of China (No.)and Shanghai Committee of Science and Technology (No.0).

Appendix A.Supp lementary d ata

Supplementary data associated w ith this article can be found,in the online version,at

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