Study Of A Chemiluminescence Biosensor For Detecting Pesticide Residues By Immobilizing Acetylcholinesterase

Organophosphorus and carbamates pesticides are widely used in agriculture due to their high insecticidal activity. However, due to the large seal of their application, the residues of organophosphorus and carbamates pesticides had brought serious environment pollution which became a greater and greater threaten to the health of human beings. At present, some sensitive and reliable detection methods have been used in practical application, such as liquid chromatography (LC), gas chromatography (GC), mass spectrometry (MS) or combinations thereof. However, these methods are need professional operators and expensive instruments, and were had to satisfy the increasing requirements of the online detection. As a comparison, biosensors provided a promising way to detect organophosphorus and carbamates pesticides which were both rapid and simple.This study applied acetylcholinesterase (AChE) of muscle of crucian carp from freshwater as a biosensor component, luminescence analyzer as detector, and continuous-flow analysis was adopted to determine organophosphorus and carbamates pesticides in the luminol-potassium ferricyanide system. The following are the process and findings of this research:1、AChE of brain and muscle of crucian carp from freshwater were used in this study as enzyme source, inhibitors were phoxime, dimethoate, dichlorvos, chlorpyrifos, carbosulfan, carbofuran, The concentration and inhibition time of these pesticides on brain and muscle AChE activity were analyzed,50% inhibition concentration (IC50) values was calculated. Results show that these pesticides all have strongly inhibition to crucian carp brain and muscle AChE activity, and there is an obvious dose-effect relationship between the activity inhibition and the concentration of pesticides. The inhibition of carbosulfan, carbofuran and dichlorvos is definitely than others. The time of pesticides inhibition rates reached the maximum on crucian carp brain and muscle were 10 min and 12 min. Thus, there’s no significant difference regarding to the sensitivity of brain and muscle AChE to pesticides, and considering for a wide source of raw materials of muscle AChE. Therefore, we will choose small carp as resource of enzyme to biosensor in the later research.2、AChE of muscle of crucian carp was purified by combination of Polyethylene Glycol (PEG) fractional precipitation and ammonium sulfate salting-out, The optimum conditions to immobilize AChE and the characters of immobilizing AChE are studied. The results show that the purified AChE had a specific activity of 2329.95μmol/min/g.the recovery was 48.36%, and a 41.37-fold-purification was achieved. The process of immobilization was as follows:0.1 g of CNBr-activated Sepharose 4B was swelled by 1 mmol/L of HC1, and mixed with AChE solution with the activity of 7 U, then, the mixture was shaked at a speed of 110r/min for 10 hours at 4℃. The immobilized enzyme produced by this method had a high activity recovery of 75%, and had a more excellent adaptability to pH and temperature than the free enzyme, and the AChE structural was changed small.3、The aim of the study was to develop a chemiluminescence (CL) biosensor for detecting organophosphorus and carbamates pesticides. A continuous-flow analysis was adopted to determine pesticides in the luminol-potassium ferricyanide system. The concentrations of pesticides were monitored by measuring thiocholine by CL detector. The inhibited enzyme was reactivated by lmM pyridine-2-aldoxime methiodide (2-PAM). The biosensor was optimized to achieve the highest sensitivity and the shortest time of analysis, different solution concentration (potassium ferricyanide, luminol), different pH, flow rate and impact time (inhibition time and reactivation time) were tested for this purpose. Under optimum conditions, the detection limits were found to be 0.070 mg/kg for dichlorvos,0.009 mg/kg for chlorpyrifos and 0.068 mg/kg for carbosulfan.To further demonstrate the applicability of the AChE-based CL biosensor, the recovery and reproducibility test was studied by adding pesticides into lake water, vegetable and fruit samples. The results display the biosensor has good recovery (between 75% and 105%) and reproducibility (RSD< 10%, n=7). The enzymatic reactor can be stored more than 2 months. The relatively CL intensity decrease about 24.63%.In addition, the principle of biosensor was discussed to a certain extent.