| Electrochemical biosensor has been widely concerned by researchers for its simple and fast operation.In recent years,with the continuous breakthrough of nano-biotechnology,abundant nano-materials have been used as carriers in enzyme,cell,DNA molecules immobilization.In this work,biosensors based on functional nano-materials has been prepared and applied in food safety testing,which has far-reaching significance for human health.Three different electrochemical enzyme electrode sensors were constructed and applied to detect organophosphorus（OPs）residues in agricultural products.The specific content are as following:（1）Acetylcholinesterase sensor based on functional multi-wall carbon nanotubes for detection of OPs residues in agricultural productsIn this study,the MWCNTs were selected as acetylcholinesterase carrier and their surface was modified simply by introducing different functional groups（-SH,-NH2,-Cl,-OH）,different hydrophobic alkyl groups(-CH3,-（CH2）2CH3,-（CH2）7CH3,-（CH2）15CH3)and different ionic liquids（Cl-,BF4-）.Finally,compared with modifiers reported in literatures and in this study,we found that MWCNTs surface modified by chloride ionic liquid was the best carrier for AChE and the detection limit of Cl/MWCNTs/AChE/GCE was 3.3×10-1111 mol L-1.At optimum reaction condition（pH7.0,AChE loading 0.25 U,Inhibition time 14 min）,Storability test indicated reactivity of Cl/MWCNTs/AChE/GCE remained above 98.5%within two weeks.For Actual sample detection,the recoveries of Cl/MWCNTs/AChE/GCE were found to be between 90.0%and 104.0%.These results demonstrated novel biosensors could act as device of high sensitivity for accurate and fast detection of OPs.（2）Acetylcholinesterase sensor based on functional MWCNTs and horseradish peroxidase for detection of OPs residues in agricultural productsCl/MWCNTs,the optimized material in part I,has been chosen as the matrix.acetylcholinesterase and horseradish peroxidase（HRP）were added in matrix.HRP has catalytic oxidation performance,Cl/MWCNTs has excellent electronic transfer performance,and novel AChE biosensor is used for the detection of OPs residue in agricultural products.The results showed that within the range of 1.0×10-11-1.0×10-7mol/L,the inhibition rate of the biosensor presented an obvious linear relationship with the concentration of OPs,and the detection limit was 4.5×10-12 mol/L.Test results showed the enzyme electrode sensor has good sensitivity.（3）Acetylcholinesterase sensor based on functional MWCNTs and bionic enzyme iron porphyrin for detection of OPs residues in agricultural productsThe use of HRP makes the sensor more sensitive,but HRP has poor stability due to nature of enzyme protein.Synthetic ferroporphyrin（FePP）is the active center of HRP,which also has similar catalytic oxidation performance with HRP.FePP has stronger stability and could been used to prepare AChE biosensor instead of HRP,which is expected to improve stability of the sensor.Based on the advantages of MWCNTs and ionic liquid,a novel and ultrasensitive bienzyme electrode（Cl/FePP-MWCNTs/AChE/GCE）including AChE and mimetic oxidase Iron-porphyrin（FePP）has been prepared for OPs detection in agricultural products.Specially,mimetic oxidase FePP was bound covalently onto AChE carrier via ionic liquid.Ultrasensitive and stable bi-enzyme electrode was obtained.At optimal condition,the detection limit of novel biosensor was 3.2×10-11 mol/L in the range of 1.0×10-10 to 1.0×10-7 mol/L.Practical sample analysis test showed that Cl/FePP-MWCNTs/AChE/GCE was recovered up to 100%.RSD was kept within3.0%after continuously detecting five times in repeatability test.After 5 weeks stored at-4 oC,the oxidation value still maintained 97.8%of the original current value.Enzymatic kinetic parameter（Km）of Cl/FePP-MWCNTs/AChE/GCE was 1.56mmol/L measured by chronoamperometry.It was deduced that AChE immobilized on Cl/FePP-MWCNTs/GCE has better affinity to substrate.The proposed biosensor exhibited excellent stability and sensitivity. |
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