Study On Highly Sensitive Enzymatic Nano-biosensor For Detection Of Pesticide Residue

With the long-term use of pesticides in agricultural products, excessive pesticide residues has caused considerable hazard on ecological environment and health of human. Organophosphorus and carbamate pesticides are two common pesticides which are still widely used in agriculture production. They can inhibit cholinesterase activity in body and cause poisoning symptom, even endanger the life of human being. So pesticide residues in food have been paid more attention over the world. Now, people would pay growing awareness of food safety, since it's extremely urgent to strengthen the detection of pesticide residues.Traditionally, the widely used methods for determination of organophosphorus pesticides are liquid/gas chromatography, mass chromatography and spectroscopy, these methods are sensitive and allow discrimination among different organophosphorus compounds but they are expensive and require a long time not to satisfy the need of fast detection. So research a rapid, sensitive and convenient method for detection pesticide residues is our imperative needs, it will play an important role in prevention high pesticide residues farm products from the marketplace and to reduce the incidents of food safety. Enzymatic biosensors have emerged as an ultra sensitive, selective cost-effective and rapid technique for pesticides residue analysis in environmental monitoring, food and quality control.In recent years, it has been given serious attention and extensive research, becoming important trends for detection pesticide residues.The acetylcholinesterase biosensors are applied to detect organophosphorus pesticides widely. The acetylcholinesterase can catalyze the substance to hydrolyze selectively. And the activity of acetylcholinesterase can be restrained by the organophosphorus pesticides. We make use of the characteristic to design the biosensors to detect the content of organophosphorus pesticides. In order to improve the sensitivity of the biosensor, the electrochemical catalyst usually be used in the preparation of the biosensor.Due to their special physical and chemical properties, nanosturctured materials can activate while active electrode surface, and promote the direct electron transfer between the active center in biomolecule and the electrode surface. Therefore, the application of nano-technology on electrochemical analysis of biological molecules is a promising area and conducive to the establishment of some new theories, new technologies and new methods.In this paper, we will propose a simple and efficient method for detection trace pesticide residues based on immobilization of AChE on nanosturctured materials modified electrode, which called electrochemical biosensor for screening of organophosphorus and carbamate pesticides. In this thesis, the novel biosensors were constructed based on nano-ZnO, nano-SiO2 and multi-walled carbon nanotubes. The electrochemical properties and catalytic effects were studied. The main contents and results are summarized as follows:(1) This experiment is mainly for the extraction of the crude enzyme solution. Acetylcholinesterase (AChE) from cephalothorax of Martianus dermestoides Chevrolat., was separated from the crude extract and purified to electrophoretic homogeneity by Sephadex G-200 chromatography. Then the properties of crude extract and purified enzyme were studied. The results show that the acetylcholinesterase from cephalothorax of Martianus dermestoides Chevrolat. optimum conditions for the extraction is,2.0%Trition X-100, pH 8.0, the ratio of material and extract 1:5, the extraction for 10 h at 4℃. The crude enzyme was purified 52.76-fold. And the content of total protein was 2.37 mg. The specific activity of enzyme was 13.19 OD/(min/mg). The 85.67% of activity recovery was obtained. The Molecular weight of the purified enzyme was 78.6 KDa, measured by SDS-PAGE. Studies on the properties of the AChE showed that the optimum temperature of the enzyme was 36℃and the exhibited optimum inhibition time about 35 min. The inhibition efficiency of dichlorvos was a linear function of its concentration from 0.2 to 2 and 2 to 10.00μM. The linearization equation were I%=(18.38c+11.25)% and I%=(1.53c+51.66)%. The detection limit was calculated to be 1.58±0.37μg/L(2) The formulation was optimized on the basis of orthogonal design and its entrapment efficiency was performed by the protamine sedimentation method. The film-evaporation combining with freeze-thawing method was used to prepare AChE liposome. The optimal conditions were found to be that the ratio of (cholesterol+egg phospholipid) and AChE was 1:2, the times of freeze-thawing was 30 times, the rotate speed was 150 rpm, the ratio of cholesterol and egg phospholipid was 2:1. The average activity entrapment efficiency of the optimized AChE liposomes was 89.5%. AChE liposomes had a mean diameter of 7.3±0.85μm and 85% (by volume) of the microspheres were below 7μm and a zeta potential of-34.7 mV. AChE liposomes bioreactor compared to the same content of the free enzyme has high stability. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The inhibition efficiencies of dichlorvos and sevin were a linear function of their concentrations. The detection limit was calculated to be 1.13±0.29μg/L and 1.36±0.35μg/L, respectively.(3) A three-electrode analysis system linked to LK98 electrochemical analyzer was used as a detection system. The results showed that the GCE-(CS/ALB)5 Could improve the catalytic current response to ATChCl significantly with the increased number of the multilayer films. The optimum assembly number was 5. The inhibition efficiency of dimethoate was a linear function of its concentration from 0.25 to 1.5 and 1.75 to 10.00μM. The linearization equation were I%= (24.45c+12.35)% and I%= (1.86c+58.76)%, with the correlation coefficients squre of 0.9981 and 0.9914, respectively. The detection limit was calculated to be 0.86±0.098μg/L.(4) Sodium silicane and anmonium chbride were used m prepare nanometer SiO2. The effects of concentration of sodium silicane, volume ratio of ethanol to water and pH value on specific surface area of SiO2 powder were investigated. The nanometer SiO2 was characterized by SEM and XRD. The SiO2 had a specific surface area of 297.3 m2/g. The results showed that SiO2 which were obtained by association had a mean length of 115.5±1.86 nm and a zeta potential of-42.5 mV. The optimum assembly number was 6. The GCE-(ALB/SiO2)6 inhibition efficiency of dimethoate was a linear function of its concentration. The developed GCE-(ALB/SiO2)6 exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.(5) Multiwalled carbon nanotubes (MWNTs) were treated in 1:3 concentrated nitric-sulfuric acid to cut them into short tubes and to create carboxyl groups at their ends. Homogeneous multilayer films of the shortened MWNTs were assembled by a layer-by-layer method, based on electrostatic interaction of positively charged cationic polyelectrolyte chitosan (CS) and the negatively charged and MWNTs. The optimum assembly number was 6. The GCE-(ALB/MWNTs)6 inhibition efficiency of dimethoate was a linear function of its concentration. The detection limit was calculated to be 0.68±0.076μg/L.(6) The main content of this chapter was to build the chemically modified electrode for the detection of pesticide residues based on (ALB/ZNO)n multilayer films.ZnO nanoparticles intermingled with Al(Al-ZnO) were gained by the sol-gel method The slid solution structure of Al-ZnO was confirmed by X-ray diffraction (XRD)and energy dispersive spectroscopy (EDS). The optimization experimental conditions were calciming temperature of 1200℃, the ratio of water and alcohol of 1.5, Zn(CH3COO)2 concentration of 1.5 mol/L, concentration of 2 mol/L and the mass fraction of Al3+adulteration of 1.5%. The optimum assembly number of (ALB/ZNO)n was 5. GCE-(ALB/ZnO)5 showed the good optical properties. The Al-ZNO-modified electrode had a higher response current with UV and visible light. The GCE-(ALB/ZNO)5 inhibition efficiency of dimethoate was a linear function of its concentration. The detection limit was calculated to be 0.76±0.087μg/L.(7) In this experiment, the nano-ZnO, MWNTs and nano-SiO2 which were through a combination of ordered nano-composite structure constructed enzyme biosensor. We got a superior performance electrochemical biosensor based on (ALB/MWNTs/SiO2/ZnO)4 film by re-optimize their working conditions. The optimal conditions were found to be that the total reaction volume of 10 mL, the reaction time of substrate 15 min, system temperature of 36℃, accompanied by UV and visible light radiation, the reaction system pH value 7.4, inhibition of pesticide samples 12 min, added 1.25 mM substrate, the reaction time 15 min. The GCE-(ALB/MWNTs/SiO2/ZnO)4 inhibition efficiency of dimethoate was a linear function of its concentration. The detection limit was calculated to be 0.53±0.096μg/L.(8) The anti-interference ability and reactivation of AChE biosensors were examinated, the characteristics of both were a direct impact on the practical application of the biosensor. The AChE biosensors were tested to study the influence of heavy metal ions, pesticides and other compounds on the corresponding enzyme. It has finally appeared that heavy metal ions and pesticides gave no significant inhibition. For ascorbic acid, the experiments showed that biosensors are quite sensitive to it. In the present study, enzyme reactivations by three oximes were explored for reactivation of the enzyme for repeated use.2-PAM was found to be a more efficient reactivator under repeated use, retaining more than 80% of initial activity after 6 reuses.(9) The electrochemical responses of 8 pesticides were detected with enzyme nano-biosensors. The results showed that the biosensor inhibition efficiencies of 8 kinds of pesticides were a linear function of their concentrations and with the low detection limit. Using acetylcholinesterase biosensor technology, taking apples, cucumbers and cabbages examples, the organophosphorus pesticide residues were determined with standard addition method. AChE had shown good accuracy, reproducibility and accuracy. It could be used for wild testing, and did not require tedious sample preparation process. It met the rapidly testing needs.