| Escherichia coli (E. coli), a typical inhabitant of the human intestinal tract, spread abroad in natural environment and contaminate water supplies and recreational waters. Most of E. coli can not cause diseases, while some of them produce enterotoxin and are the major cause of infection outbreaks with serious consequences. In particular, the serotype 0157 associates with several human diseases including diarrhoea, hemorrhagic colitis and hemolytic-uremic syndrome. E. coli is routinely used as an indicator to monitor potential enteric pathogen contamination of waters. Therefore, it's important to develop a rapid detection method of E. coli.Conventional methods for detection of E. coli mostly include multiple-tube fermentation and membrane filter. But these methods were not suitable for rapid detection of contamination because of their long detection period and complex operation. Besides, fluorescence assay, chemiluminescence assay, immunoassay, polymerase chain reaction method and electrochemical method have been developed. These methods shortened the detection period and simplified the detection procedure, but most of them can't get high sensitivity and specificity as conventional methods.Recently, more and more nanostructure materials were applied in analytical chemistry. A MWNTs/Nafion/GCE biosensor was developed based on nanostructure materials to detect the concentration of E. coli, with detection limit of 10 cfu/mL and detection time of 5 h. Due to the good biocompatibility of the nanomaterials, a rapid, specific and sensitive method for assay of E. coli using Au and CdS nanoparticles as antibody labels in combination with immunomagnetic separation technique was proposed. The proposed method was applied to detect the E. coli with low detection limit of 30 cfu/mL, and the total detection time was about 2 h. The dissertation includes four sections.Chapter one:IntroductionBasic knowledge on E. coli was introduced including biological feature, pathogenicity and sanitation standard. Then, Conventional methods and some novel methods for detection of E. coli were demonstrated. What'more, excellent properties, preparation and characterization of nanostructure materials and the appplication of nanostructure materials on rapid detection of E. coli were presented. Chapter two:Rapid amperometric detection of coliforms based on MWNTs/Nafion composite film modified glass carbon electrodeMulti-wall carbon nanotubes (MWNTs)/Nafion modified glassy carbon electrode (GCE) was fabricated for the rapid amperometric assay of E. coli. Due to the cation-exchange capacity of Nafion and the electrocatalytic ability of MWNTs, the detection sensitivity of PAP was improved and the detection time of E. coli was shortened. Under the optimized experiment conditions,10 cfu/mL E. coli could be detected after 4.5 h enrichment. The electrochemical sensor was further used to detect E. coli in real samples, and the results were consistent with that obtained by the plated count method.Chapter three:Gold Nanolabels for Enhanced Electrochemical Immunoanalysis of Escherichia coliA novel nanolabel based on gold nanoparticles modified with anti-E. coli peroxidase (HRP)-conjugated antibody has been developed. It was used for enhanced electrochemical immunoanalysis of E. coli. After the immuno-reaction, gold nanolabel, immunoparamagnetic bead (IMB) and E. coli formed a sandwich-type immunocomplex. 3,3',5,5'-tetramethylbenzidine (TMB) was used as substrate for HRP in the presence of H2O2 and the enzyme activity of HRP was measured by electrochemical detection coupled with flow injection assay (FIA). The concentration of E. coli could be confirmed by amperometric response, which was in proportion to the quantity of HRP on the sandwich-type immunocomplex. Gold nanoparticles were used as carriers of antibodies resulting in the increase of antibodies attached to E. coli. Therefore, the amperometric signals were enhanced and the detection sensitivity of E. coli was improved greatly. It showed a good linear relationship between the amperometric responses and logarithmic value of E. coli concentrations ranging from 1.0×102 to 5.0×104 cfu/mL, with detection limit of 50 cfu/mL. By introducing a pre-enrichment step, a lower detection limit and wider detection range could be obtained. The rapid detection could be finished within 1 h, shorter than other methods.Chapter four:CdS Nanoparticle Labels for Enhanced Immunomagentic Electrochemical Rapid detection for Escherichia coli A new magnetic electrochemical immunoassay has been developed as a tool for rapid detection of Escherichia coli (E. coli). The method uniquely combined use of immunomagnetic beads (IMBs) for target capturing/enrichment coliforms and semiconductor quantum dots (QDs), which were successfully conjugated to an anti-E. coli antibody via a cross-linking reaction, as tracers for ultrasensitive square wave voltammetry (SWV) analysis. The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on.a glass carbon electrode. The new immunoassay detection capability is coupled with high sensitivity and absence of nonspecific interactions. It showed a good linear relationship between the amperometric responses and logarithmic value of E. coli concentration ranging from 5.0×101 to 1.0×105 cfu/mL. The proposed technique was applied to measure E. coli with a low detection limit of 30 cfu/mL and the overall analysis could be completed within 2 h.
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