| Pesticides play an important role in the improvement of agricultural food production. With the banning of organochlorine pesticides, organophosphorus pesticides(OPs) were used extensively due to their relativily low persistence in the environment. By now, over one hundred of OPs have been registered. OPs residued in foodstuff especially in vegetables is an escalating problem that has aroused public concern over potential health hazards. Current analytical techniques such as gas chromatography(GC), are very sensitive and reliable, but are time consuming, expensive, and can't be carried out infield. Therefore, analytical devices for the rapid, sensitive, selective and reliable detection of OPs residues in vegetables, especially for on-line monitoring, is still desirable. Biosensors based on enzyme seem to be suitable for such a purpose.The study of ecotoxicology has displayed, the toxicity of OPs is commonly attributed to their inhibition effects on acetylcholinesterase (AChE, EC 3.1.1.7) in animals, and the degree of inhibition is dependent on the concentration of OPs. So, the measurement of inhibition of AChE activity provides the analytical basis for AChE-based biosensor to monitor OPs.AChE from various biological materials has different sensitivity to OPs obviously. In order to improve the response of AChE-based biosensor to OPs, it is necessary to select a sensitive AChE among different organisms. In this research, the comparative study for the sensitivity of acetylcholinesterase(AChE) from three biological materials (electric organ of Electrophorus electricus, brain of Scomberomorus niphonius, and body of Musca domestical to 4 OPs (malathion, parathion-methyl, phoxim and chlorpyrifos) was conducted in the same conditions. The results showed that: the sensitivity of AChE to malathion and parathion-methyl was in the order as follows: Electrophorus electricus > Scomberomorus niphonius > Musca domestica; While, for the sensitivity to phoxim and chlorpyrifos, there is another order as follows: Musca domestica > Electrophorus electricus > Scomberomorus niphonius. By comprehensive consideration basing on these results above, the AChE from electric organ of Electrophorus electricus has been considered to be the better enzyme for indicating these four OPs.The immobilization of enzyme is a key step for the preparation of biosensor. The quality of immobilized enzyme may affect the stablity, sensitivity, selectivity and the application of biosensor directly. Two methods were used for the immobilization of AChE from electric organ of Electrophorus electricus, one was covalent combination with CNBr activated sepharose 4B gel as a carrier, another one was crosslink by glutaraldehyde with Aminopropyl-controlled porous glass (CPG) as a carrier. It was proved that the activity recovery of the immobilized AChE by crosslink method was lowered by 10% than that by covalent combination. There was a less difference of preservation time between the two immobilized enzymes. But for the life-span, crosslink method was better than covalent combination: the enzyme immobilized by this method could maintain a stable activity during continous use for 28 times, with a good reproducibility (RSD=0.96%). Therefore, crosslink method with aminopropyl-CPG as a carrierwas selected to prepare the immobilized enzyme in the following research.The dose-effect relationship between OPs and the immobilized AChE is essential for the accurate detection of OPs in samples. A flow injection biosensor was constructed with the immobilized AChE as a identification element and pH eletrode as a transducer. The suitable working conditions for this biosensor were listed as follows: the initial activity of enzyme used for immobilization was 20u; the concentration of substrate solution was 20g/L; both the blank solution and carrier solution was phosphate buffer of 1mmol/L (pH=7.5), the inhibition time was 10 min; and the flow rate of main channel is 0.45mL/min. On these conditions, the change of enzyme activity with the concentration of OPs was determined. Thus, the concentration-activity inhibition curves for malathion, parathion-methyl, phoxim and chlorpyrifos (after preoxidization treatment) in a concentration range of 10~500μg/L, were obtained respectively. In order to increase the sensitivity of the biosensor, NaClO was used for the preoxidation of OPs. For the For the reactivation of the immobilized enztme, a 2-PAM solution was used.For the programmed work during sample dection, a computer with special software was connected to the biosensor. The mornitoring process consisted of 5 steps: sample preoxidation, baseline stabilization, appearence of initial activity peak before sample addition, appearence of inhibition activity peak after sample addition, the calculation and display of OPs content. A sample detection was completed within 30 minutes. The detection limit for malathion, parathion-methyl, phoxim and chlorpyrifo (after preoxidization treatment) in phosphate buffer of lmmol/L (pH 7.5) were 10.4μg/L, 14.3μg/L, 28.5μg/L and 9.5μg/L respectively. That is to say, when extration rate (ratio of sample weight and solvent volume for extraction ) was 1, the sensitivity of biosensor for these OPs in vegetable samples would be satisfied with the request of national standard for OPs residue control (GB14868-94, GB 14874-94, etc).The developed biosensor was applied for mornitoring the extration solution from rape samples, containing 100Mg/kg of OPs mentioned above, with phosphate buffer containing methanol of 1% as a extrating agent, the detection values obtained had relative errors of 11.4%, +22.5%, -30.0% and +86.1% for malathion, chlorpyrifo, parathion-methyl and phoxim respectively. Therefore, the detector developed in this study had a better accuracy for the former three OPs.In brief, the detector developed in this study could be applied for rapid, simple, sensititive, accurate and easily automobile determination of OPs, such as malathion, chlorpyrifo and parathion-methyl. It will be beneficial for the control of OPs residue in vegetables. |
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