Immobilization of acetylcholinesterase on f(2)

The 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 centrifugation.

The SBA-15 and NH2-SBA-15 w ere characterized by XRD,TG-DSC,FT-IR,XPS,TEM and N2adsorption-desorption isotherms.

Mesoporous 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(□),SAC

The 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 immobilization.

In 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 condition.

Table 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 9.0×10-4

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