Immobilization of acetylcholinesterase on f(4)

[15],J.M.Wang,S.Wu,,T.Yang,(2013)1097–1104.

[16]X.S.Guo,,Q.Cai,T.Shen,S.M.Zhu,(2013)15–23.

[17],T.Itoh,T.Sumiya,,M.Ono,(2009)443–448.

[18],,S.Bhand,R.Mu?oz,,(2012)56–61.

[19]H.P.Mao,Y.T.Yan,N.Hao,et al., (2017)239–248.

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[23]T.Itoh,R.Ishii,,et al., (2009)183–187.

[24],,H.Yari,A.Ghaedi,(2016)265–270.

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[38],X.S.Zhao, 107(2003)–.

[39],,,,M.Sastry,(2005)5115–5121.

[40]M.Saleem,M.Ra fiq,S.Y.Seo,K.H.Lee,(2016)e00311.

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[42]H.Tümtürk,F.Sahin,,(2007)141–145.

[43]J.Marty,K.Sode,I.Karube,Electroanalytical 4(1992)249–252.

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 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 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 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 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 immobilization of ACh E on SBA-15 or NH2-SBA-15 w as performed in tw o 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: 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 experiment process of vegetables w as modi fied by the literature[34]as follow s:Fresh vegetables w ere chopped after w ashing,then 2.0 g of each sample w as sprayed w ith 1 m L 1×10-4ppm trichlorfon and 2×10-3ppm carbaryl,respectively,and keep at room temperatures for 4 h before sample w as extracted by adding 9 m L of 0.1 mol/L PBS of 8.0 and treated w ith ultrasonic for 5 min,then tested by Microplate Reader in NAC after the suspension w as separated by SBA-15 and NH2-SBA-15 w ere characterized by XRD,TG-DSC,FT-IR,XPS,TEM and N2adsorption-desorption silica is an easily fabricated material that has a tremendously high ratio of surface area to volume,making it an ideal material for enzyme XRD patterns of the SBA-15 and NH2-SBA-15 materials in Fig.S1(Supporting information)show the presence of the three re flection peaksat 2θ=0.8°–2°corresponding to 100,110 and 200 planes,w hich indicates that the samples have a highly ordered hexagonal P6 mm square ratio of 2θis about 1:3:4 and the presented structure is consistent w ith the previous report[35].The textural properties including BET surface area,pore diameter and pore volume of NH2-SBA-15 together w ith SBA-15 w ere calculated by N2adsorption-desorption isotherms,and the results w ere show n in Table S1(Supporting information).The pore diameter of the materials is larger than the molecular dimensions of ACh E[36],show ing that the mesoporous materials are suitable to immobilize ACh E.In addition,the sample w as set in thermal analysis apparatus and TG-DSC curve(Fig.S2 in Supporting information)w as amounts of amino group into the pores of SBA-15 w ere estimated to be 2.76 mmol/g.Fig.S3(Supporting information)show s the FT-IR spectra of the support materials and illustrated that amino had been modi fied into SBA-15[37,38].The XPSand TEM resultsfurther con firmed the mesoporous structure of SBA-15 and NH2-SBA-15(Figs.S4–S6 in Supporting information).Immobilization of ACh E on NH2-SBA-15 w as conducted by the method of covalent coupling or adsorption-crosslinking as show n in Scheme 1.Scheme of ACh E on NH2-SBA-15 mesoporous molecular sieve by covalent coupling(A)or adsorption-crosslinking(B)method. residual activity of different immobilized ACh E stored at 4°C and 25°C after 60 :NACNCC(□),SACThe effect of enzyme/material ratio on the immobilization w as investigated by varying the concentration of ACh E from 0.5 mg/m L to 35 mg/m L in the presence of support material concentration(10 mg/m L),and three kinds of materials w ere tested,including SBA-15(pore diameter is 6.1 nm or 12.5 nm)and NH2-SBA-15(pore diameter is 10.2 nm).For SBA-15,w ith the increasing of the enzyme/material ratio,the amount of immobilized enzyme increased linearly until it reached the dynamic balance betw een adsorption and maximum adsorption amount of AChE reached 85 mg/g(protein/material)for SBA-15(6.1 nm),250 mg/g(protein/material)for SBA-15(12.5 nm)and 55 mg/g(protein/material)for NH2-SBA-15(10.2 nm),w hich meant that bigger pore diameter of SBA-15 w as more suitable for enzyme possible explanation is that ACh E could be entrapped into both inside of the channel and the surface of the materials and the functionalized SBA-15 restricted the protein coming into the ever,the protein immobilization ef ficiency of ACh E on NH2-SBA-15(＞95%)w as much higher than that of SBA-15(60%).NH2-SBA-15 show ed the highest immobilization ef ficiency,being very likely due to the increase of electrostatic interactions,and there w as no enzyme addition,the speci fic enzyme activity reached the maximal value at low concentration of ACh E addition(1 mg/m L).And the highest speci fic activity of immobilized ACh E w as 30%-40%higher than that of the free one,pointing tow ards a higher af finity betw een all the active-site groups and ,1 mg/m L of ACh E and 10 mg/m L NH2-SBA-15 w ere chosen as the optimal enzyme amount and carrier for order to improve the properties of immobilized ACh E,adding certain stabilizing agents is necessary,like BSA,w hich was frequently used[39].Fig.S7(Supporting information)demonstrated the effects of BSA concentrations on the activity and stability of ACh E-NH2-SBA-15 prepared according to NAC,immobilized enzyme test was performed in 0.5 mol/L PBS solution of p H 8.0 at 50°C.It can be clearly seen that high residual activity of immobilized enzyme w as obtained at 0.4%of BSA concentration,but quickly declined as the concentration ,1 mg/m L ACh E and 0.4%BSA w ere added in turn on the surface of 10 mg/m L NH2-SBA-15 by electrostatic adsorption for 30 min independently,then glutaraldehyde w as added for cross-linking w ith 2 h,is regarded as the optimal immobilization 1 Linear range and LOD of immobilized ACh E and free ACh E for the detection of carbaryl and Trichlorfon Linear range(ppm) LOD(ppm) Linear range(ppm) LOD(ppm)ACh E-NH2-SBA-15 1.0× 7.6×10-41.0×10-5-1.0 2.4×10-6Free AChE 5.0×10-3-5.0 2.0×10-31.0×10-3-1.0 9.0×10-4Table 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× 4.76The 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 re 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 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 ledgmentsThis 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 ataSupplementary data associated w ith this article can be found,in the online version,at References[1]A.Crew,,N.Byrd,,J.P.Hart,(2011)2847–2851.[2]X.Sun,X.Y.Wang,(2010)2611–2614.[3]Y.Q.Miao,N.Y.He,J.J.Zhu,(2010)5216–5234.[4],,R.Mu?oz,A.Hayzt,J.Marty,(2015)491–496.[5]Z.M.Liu,Y.F.Jing,Z.L.Wang,H.J.Zhan,Q.Shen,(2013)531–538.[6],K.L.Fan,Y.Pan,et al.,(2013)308–312.[7],Y.N.Cai,Z.L.Qi,L.Lu,Y.X.Qian,(2013)8455–8461.[8]H.C.Li,Y.X.Guo,L.H.Xiao,B.Chen,Analyst 139(2014)285–289.[9],,A.S.Cans,Langmuir 30(2014)–.[10]D.B.Liu,W.W.Chen,J.H.Wei,et al.,(2012)4185–4191.[11]C.P.Li,S.M.Fan,C.Y.Yin,et al., 6(2014)1914–1921.[12],S.A.Alex,,,(2016)–.[13],J.Chen,Y.T.Wang,et al., (2014)904–910.[14],L.G.Shan,Z.R.Tian,et al.,(2008)592–594.[15],J.M.Wang,S.Wu,,T.Yang,(2013)1097–1104.[16]X.S.Guo,,Q.Cai,T.Shen,S.M.Zhu,(2013)15–23.[17],T.Itoh,T.Sumiya,,M.Ono,(2009)443–448.[18],,S.Bhand,R.Mu?oz,,(2012)56–61.[19]H.P.Mao,Y.T.Yan,N.Hao,et al., (2017)239–248.[20],,,et al.,(2015)209–217.[21]J.Fan,C.Z.Yu,F.Gao,et al.,(2003)3146–3150.[22]J.L.Blin,C.Cérardin,,et al.,(2005)1479–1486.[23]T.Itoh,R.Ishii,,et al., (2009)183–187.[24],,H.Yari,A.Ghaedi,(2016)265–270.[25]X.Y.Sun,P.Z.Li,B.Ai,Y.B.Wang,(2016)139–144.[26],B.Li,T.Sasaki,et al.,(2000)3301–3305.[27]Z.X.Ao,J.H.Hui,W.X.Feng,et al.,(2009)3023–3028.[28]M.M.Wan,W.G.Lin,L.Gao,H.C.Gu,J.H.Zhu,(2012)497–503.[29]C.H.Lei,Y.Shin,J.Liu,,(2002)–.[30],,,B-Enzym.15(2001)81–92.[31],Y.Geiger,G.Goobes,(2014)9031–9038.[32]J.B.Kim,,P.Wang,(2006)1017–1026.[33]D.Y.Zhao,P.D.Yang,N.Melosh,et al.,(1998)1380–1385.[34],Q.C.Xu,Y.M.Guo,X.Sun,X.Y.Wang,RSC Adv.6(2016)–.[35],Y.Zou,Y.Shen,et al.,(2014)1541–1549.[36]ández,,,et al.,(2007)444–448.[37]K.Kannan,,B-Enzym.56(2009)34–40.[38],X.S.Zhao, 107(2003)–.[39],,,,M.Sastry,(2005)5115–5121.[40]M.Saleem,M.Ra fiq,S.Y.Seo,K.H.Lee,(2016)e00311.[41]T.Itoh,,,et al.,Analyst 139(2014)4654–4660.[42]H.Tümtürk,F.Sahin,,(2007)141–145.[43]J.Marty,K.Sode,I.Karube,Electroanalytical 4(1992)249–252.

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