| Escherichia coli(E.coli),a typical inhabitant of the human intestinal tract,spreads abroad in natural environment and contaminates water supplies and recreational waters.It can be a causative agent of intestinal and extra-intestinal infections,may disseminate throughout the body,and has been implicated in urinary tract infection,sepsis,meningitis and gastro-intestinal infections.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 they can't get high sensitivity and specificity as conventional methods.Recently,more and more nanostructure materials were applied in analytical chemistry.A Tyr/MWNTs-Chit/GCE biosensor and Tyr/Fe3O4 MNPs-CNTs/GCE biosensor was developed based on nanostructure materials detect the concentration of coliforms,with detection limit of 10 cfu/mL and detection time of 5 h.Then,a rapid, specific and sensitive method for assay of Escherichia coli(E.coli)using biofunctional magnetic nanoparticles(BMNPs)in combination with adenosine triphosphate(ATP) bioluminescence was proposed.The proposed method was applied to detect the E.coli inoculated into pasteurized milk with low detection limit of 20 cfu/mL,and the total detection time was about 1 h.The dissertation includes three 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 were presented,and the appplication of nanostructure materials on rapid detection of E.coli. Chapter two:Fabrication of Tyrosinase Biosensor Based on Multiwalled Carbon Nanotubes-Chitosan Compostie and Its Application to Rapid Determination of ColiformsA tyrosinase(Tyr)biosensor was fabricated by immobilizing Tyr to a multiwalled carbon nanotubes(MWNTs)-chitosan(Chit)composite modified glassy carbon electrode (GCE).The MWNTs-Chit composite film provided a biocompatible microenvironment for Tyr,and the MWNTs possessed excellent inherent conductivity to enhance the electron transfer rate.The Tyr/MWNTs-Chit/GCE biosensor showed high sensitivity(412μA mM-1),broad linear response range from 1.0×10-8to 2.8×10-5M,low detection limit(5.0 nM)and good stability(remained 93%after 10 days)for determination of phenol.The biosensor was further applied to rapid detection of the coliforms.The detection principle was thatβ-D-galactosidase,an indicator of coliforms,catalyzed hydrolysis of phenol-β-D-galactopyranoside(PG)to produce phenol,and the concentration of phenol was electrochemically determined by the biosensor.The current response was proportional to the quantity of coliforms,represented by Escherichia coli(E. coli)in this work,and E.coli could be detected as low as 10 cfu/mL after 5.0 h of incubation.Chapter three:Amperometric Tyrosinase Biosensor Based on Fe3O4 Nanoparticles-Coated Carbon Nanotubes Nanocomposite for Rapid Detection of Coliforms with Flow Injection AssayA tyrosinase(Tyr)biosensor was developed based on Fe3O4 magnetic nanoparticles (MNPs)-coated carbon nanotubes(CNTs)nanocomposite and further applied to detect the concentration of coliforms with flow injection assay(FIA)system.Negatively charged MNPs were absorbed onto the surface of CNTs which were wrapped with cationic polyelectrolyte poly(dimethyldiallylammonium chloride)(PDDA).The Fe3O4 MNPs-coated CNTs nanocomposite was modified on the surface of the glassy carbon electrode(GCE),and Tyr was loaded on the modified electrode by glutaraldehyde.The immobilization matrix provided a good microenvironment for retaining the bioactivity of Tyr,and CNTs incorporated into the nanocomposite led to the improved electrochemical detection of phenol.The Tyr biosensor showed broad linear response of 1.0×10-8- 3.9×10-5M,low detection limit of 5.0×l0-9M and high sensitivity of 516 mA/M for the determination of phenol.Moreover,the biosensor integrated with a FIA system was used to monitor coliforms,represented by Escherichia coli(E.coli).The detection principle was based on determination of phenol which was produced by enzymatic reaction in the E.coli solution.Under the optimal conditions,the current responses obtained in the FIA system were proportional to the concentration of bacteria ranging from 20 to 1×105 cfu/mL with detection limit of 10 cfu/mL and the overall assay time of about 4 h.The developed biosensor with the FIA system was well suited for quick and automatic clinical diagnostics and water quality analysis.Chapter four:Combining Biofunctional Magnetic Nanoparticles and ATP Bioluminescence for Rapid Detection of Escherichia coliA rapid,specific and sensitive method for assay of Escherichia coli(E.coli)using biofunctional magnetic nanoparticles(BMNPs)in combination with adenosine triphosphate(ATP)bioluminescence was proposed.The BMNPs were fabricated by attaching a specific anti-E.coli antibody on the surface of amine-functionalized magnetic nanoparticles(about 20 nm in diameter),which were prepared through a facile one-pot strategy.The preparation procedure of the BMNPs was optimized to enhance the capture efficiency to E.coli.E.coli cells captured by the BMNPs was separated from sample simply with the assistance of an external magnet,and then detected by ATP an bioluminescence method.The optimization of ATP measurement was carried out in order to improve the detection sensitivity.The proposed method combining the BMNPs with ATP bioluminescence was applied to detect the E.coli inoculated into pasteurized milk with low detection limit of 20 cfu/mL,and the total detection time was about 1 h.Chapter five:Development of apparatus for rapid electrochemical detection of bacteriaAn apparatus for rapid electrochemical detection of bacteria was developed based on the biological feature of bacteria and electrochemistry principle.The apparatus not only detect the total bacteria in water,but also detect the concentration fo E.coli rapidly, sensitivly and accurately.Total bacteria ranging from 1.0×102 cfu/mL to 1.0×105 cfu/mL could be detected by the appratus with detection limit of 100 cfu/mL.Moreover,the appratus could detect E.coli ranging from E.coli ranging from 20 cfu/mL to 1.0×106 cfu/mL with detection limit of 20 cfu/mL.The apparatus was portable,steady,reliable and easy to use.
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