Preliminary Study On Drinking Water Endotoxin And Endotoxin Immunosensor

Endotoxin is a cell wall component of gram-negative bacteria, which is located in the outermost cell wall, lies outside the peptidoglycan on the cell wall. Live bacteria will release the endotoxin, while the cells lyse or destroyed by artificial methods. The effective part of endotoxin is lipopolysaccharide(LPS). Different species of gram-negative bacteria have the similar LPS structure which consist of three parts, the O side chain, the core polysaccharide and lipid A. Lipid A is the major toxic component of endotoxin.Endotoxin is widely spread in air,soil,water while these enviroments are closely related to the human health, and the human body are extremely sensitive to endotoxin, a very low concentration (1-5ng / kg body weight) of endotoxin can result in increased body temperature. In addition to thermal reaction, the endotoxin also cause a decrease in the number of interleukin, Shwartzman reaction, endotoxin shock, microcirculation, intravascular coagulation and so on, to adversely affect human health.At present, the major endotoxin detection methods are: rabbit pyrogen test, LAL( Limulus amoebocyte lysate) method, immunoassay, bio-sensor method.Drinking water is associated with human health.Extensive investigations for endotoxin pollutition in source water and drinking water have been made in overseas while such investigations are still in blank in China. Exploring of endotoxin levels in the production chain of drinking water would contribute to the assessment of purification effect for endotoxin and provide scientific basis for criteria formulation.Currently most endotoxin detections in China and abroad were carried out by LAL test which was regarded as the gold standard." LAL is rapid, sensitive (0.01-1ng/ml), simple-operated and widely applied. But it is not easy to distinguish between weak positive and negative, need to kill a large number of horseshoe crab, difficult to identify marginal results and highly reagent consuming. Other methods such as the rabbit pyrogen test is low sensitivie, time-consuming, and has been gradually phased out; ELISA is less time-consuming, high sensitive and specific, good reproducibile, easy to operate but highly reagent consuming and instrument demanding. In recent years biosensors were developed as new technology for rapid detection of pathogenic factors. Such detection method is less time-consuming, high sensitive and specific. The enzyme immunosensor, a particular biosensor with combination of electrochemical magnification and immunologic specificity, is not only electrochemically sensitive but also target-recognition specific. Enzyme immunoelectrode is flexiblely designed and constructed, easily to be developed a integrated and miniaturized system. The detection instrument of enzyme immunosensor is relatively simple and easy to use, less manual operation and more intelligent.Endotoxin levels in the production chain of drinking water in Wuhan have been detected and endotoxin removal effects in the water treatment have been illustrated. The electro- deposition method have been used to establish a new type of enzyme immunosensor for rapid detection of endotoxin, and its performance characteristics have been studied and discussed.PartⅠDetection of endotoxin in the procedure of drinking water treatment in Wuhan CityObjective: To investigate the pollution of endotoxin in drinking water and the endotoxin's removal effect through conventional drinking water treatment of Wuhan city in Hubei province. Methods: In Mar, 2008, water samples of Pinghumen and Zongguan drinking water plants, which sourced from the yangtze river and Han river respectively, were collected. The samples were tested by Limulus amebocyte lysate test recorded by Chinese Pharmacopoeia (2005 edition). Results: The endotoxin content of raw water of the yangtze river ranged from 98.08 to 104.08EU/ml and tap water ranged from 18.03 to 22.58EU/ml;The endotoxin concentration in the raw water of the hanjiang river ranged from 84.33 to 86.94EU/ml and tap water ranged from 40.20 to 44.44EU/ml;the removal rates of two drinking water plants were 76.98～82.68% and 47.30～53.76%respectively. Conclusion: Conventional drinking water treatment could effectively remove the endotoxin,but the content of endotoxin in finished water is still higher than that of foreign plants.PartⅡPreliminary Study on Immunosensor for EndotoxinObjective: To develop an immunosensor for endotoxin detection. Methods: The enzyme immunosensor was constructed by immobilizing monoclonal mouse IgG for endotoxin on the surface of rabbit anti-mouse IgG-HRP/ nano-gold modified glassy carbon electrode through electro-deposition , electrostatic adsorption and antigen-antibody binding reaction. The electrical response caused by capture of endotoxin on the antibody was detected by Cyclic Voltammetry and Controlled Potential Electrolysis. The optimal condition was determined by a comparative analysis of tests'results in different reaction systems,such as buffer pH, media concentration, substrate concentration, as well as reaction temperature. Results: pH 7.0 buffer, dimethylamino-methyl ferrocene concentration of 1mmol / L, H2O2 concentration of 0.2mmol / L, temperature 37℃were optimal conditions for test. It was shown that detection limit of 0.01Eu/mL, linear range of 0.01-100Eu/mL and correlation coefficient of R = 0.930 were found for the immunosensor. Conclusion: Preparation of the sensor method is simple, and relatively sensitive.