Construction Of Surface Plasmon Resonance Biosensor And Application To Rapidly Detect E. Coli O157:H7

With China’s urban and rural economic development and the improvement of people’sliving standard, the amount and type of food are increasingly rich. How to improve the qualityand safety of food becomes the growing problem. According to the statistics at home andabroad, the percentage of bacterial food poisoning is the largest in all kinds of food poisoning.Among them, the escherichia coli bacterial food poisoning is one of the most commonpathogenic bacteria and great harm to human body. Therefore, the food industry needs a quickand easy analysis method to detect the microbial contaminant of food.The novel detection technology of surface plasmon resonance （SPR） assay is known tomonitor interaction between bimoleculars real time and ascertain the kinds of reactants as wellas their concentrate without isolation and purification. SPR sensors are valuable analyticaltools with a broad range of applications from laboratory assays to study for food safety, drugdiscovery and environmental pollutant monitoring, which have the feature of high sensitivity,label free, small amount of sample and rapid speed of detection. The first surface plasmonresonance （SPR） sensor was developed in1983by Liedberg et al, since then, the research andapplication of SPR technology have shown extensive growth and gradually become the hotspot and research frontier in the biosensor field in the world.In our work, the biotin-avidin system and silver nanoparticles were applied into surfaceplasmon resonance （SPR） biosensor in order to ensure the accuracy of the test and amplify thesignal of detection. These methods, which were used to detect E. coli O157: H7, expanded thedetection limit of the surface plasmon resonance （SPR） biological sensors on the escherichiacoli bacterial. The main contents are listed as follows.1. The SPR biosensor was used to detect E. coli O157: H7directly. By using molecularself-assembly technique, the surface plasmon resonance （SPR） biosensor chip was modified.Using MPA as basement membrane, antibody was successively assembled in the sensor chip.Because of the specific binding between antigen and antibody, the combination of theescherichia coli bacterial and sensors was realized. The series of concentration E. coli O157:H7sample solution flows through sensor chip and the response signal was recorded. Theexperimental results show that, when the concentration of E. coli O157: H7is low, the response signal of surface plasmon resonance （SPR） biosensor is weak and the detectionresult is not accurate. The concentration of10⁵cfu·mL^-1can be detected.2. Based on the highly specific binding force between biotin and avidin, the bridgeconnection as well as the amplification, the biotin-avidin system was applied into SPRbiosensor. Firstly, the MPA was fixed on the surface of the SPR gold surface by self-assembly.On the basement membrane, avidin and antibody which were successively assembled wereformed. The biosensor with antibody on the surface can combine and detect E. coli O157: H7.The experimental results show that: because of the application of biotin-avidin system, themultilayer structure was formed in the sensor surface and enlarge the detection responsesignal. It is beneficial to the escherichia coli bacterial detection. Compared with the chipwithout biotin-avidin system, the response signal was increased and the detection limit wasreduced. The concentration of10⁴cfu·mL^-1can be detected.3. The silver nanoparticles were applied into SPR biosensor for bacteria detection. Firstly,the redox synthesis method was exploited to synthesize the silver nanoparticles. The silvernanoparticles was functionalized by AET and characterized by ultraviolet spectroscopy. Thefunctionalized silver nanoparticles were immobilized on gold substrates. The results indicatethat silver nanoparticles increase the surface area to volume ratio which induces an increasedsensitivity and an improved detection limit relative to bulk gold. Detection limit of10³cfu·mL^-1of E. coli bacteria has been obtained for this biosensor.