Rapid Assay For Microbe Using SPR Biosensor

This program is that the Jilin Science and Technology Office of 2002 years imburseing the international cooperative research project: "Use the SPR immunity sensor detection research of typhoid Salmonella bacterium", project serial number: 20,020,802, undertaken from the Biology and Agriculture Engineering College of Jilin University. Along with the raising of the economic development and people's living standard of our country, the kind and the quantity of food are increasingly rich, and how to raise the food quality and safety stick out increasingly. After having solved the basic need, the customers of town and country ask to eat more safety, this is the social tendency. During the food processing, store, transportation, sell, till to be eaten , the food has possibility to get biological pollution and endangers the health of human body. According to domestic and international statistics, in various bromatoxisms, the germ bromatoxism is the most. Salmonella and E.Coli are the most common pathogenic bacteria in germ bromatoxism, and endanger for human body greatly. The Centers for Disease Control and Prevention (CDC) approximates that 6,500,000 people in the United States are annually affected by pathogen in food, including 9,000 deaths. E.Coli is especially harmful to human health. With highly contagion, E.Coli is the main pathogen of infant diarrhea and seriously can lead to death. Especially E.Coli O157:H7can lead to hemorrhagic colitis (HC). Typhoid Salmonella bacterium is the most common original bacterium in food. Salmoella bacterium belongs to intestines bacillus genus, typhoid and associate typhoid can directly arouse the disease of mankind. Salmonella bacterium pollutive food can arouse the Salmonella bacterium bromatoxism Salmonella bacterium bromatoxism is the most common in germ bromatoxism, take the 42.6% ～60% of germ bromatoxism approximately according to statistics. Bromatoxism leads to urgent disease, patient centralize, thus fast accurate diagnosis is particularly important for epidemic situation control. So food industry needs an rapid and simple detection assay for the pathogen in food, especially for E.Coli and Salmonella. Now in the world, the SPR biosensor used in the research of microorganism detection is not much, and the detection limits of E.Coli and Salmonella are not high. General research can only reach the detection limit of 106 cfu/ml. Medina et al used SPR-immunochemical methods to detect E.Coli O157:H7.The direct detection of bacteria cells provided low responses. So sandwich immunoassays were utilized. This approach has produced an assay with detection limits of about 106cfu/ml for E.Coli O157:H7. Use avidin-biotin system and bind the antibody to the avidinated surface via avidin-biotin interaction for signal amplification of SPR biosensor. Thus the detection respond signal increase substantially, at the same time have raised detection limit notably. The Neutravidin we use can guarantee the accuracy of detection. To raise the sensitivity of detection, Sandwich assay has been used to amplify the detection respond .In order to increase assay sensitivity, we use sandwich assay to detect. The secondary antibody is labeled by colloid gold as second antibody to signal amplification. Use all these methods to detect E.Coli and Salmonella extend the detection limit and realized fast detection. This paper draws conclusions as below through experiments: 1. Avidin and biotin system applies to SPR biosensor detection. Avidin joints of the golden membrane surface, and to make detection microorganism become possible. The very strong affinity between avidin and biotin, also combine the antibody labeled by biotin to golden membrane surface. Finally the combination between antibody and antigen has realized the combination of microorganism and sensor, such the multilayer membrane structure forms. Thus the respond signal has been amplified, and is helpful for microorganism detection. 2.After using the avidin-biotin system ,the SPR biosensor can detect the E.Coli 0157:H7,whose concentration is 106cfu/ml. 3. Adopt the method of joining secondary antibody, that is to combine secondary antibody after the combination of bacteria and antibody, so make detection respond signal increase. The secondary antibody is immunity colloid gold .This method extends the detection limits from 106cfu/ml to101cfu/ml, and the detection time is less than 1hour.