| The timely detection of pathogens in foods and waters is a subject of greatimportance for the protection of environmental sanitation and our health.Escherichiacoli (E.coli) which are found in large numbers among the intestinal of humans andother warm-blooded animals spread abroad in natural environment.Most of E.colican not cause diseases,while part of them produces enterotoxin and are the majorcause of infection outbreaks with serious consequences.In particular,the serotypeO157 associate with several human diseases including diarrhoea,hemorrhagic colitisand hemolytic-uremic syndrome.The presence of E.coli is routinely used as anindicator to monitor potential enteric pathogen contamination of waters and foods.Therefore,it is a challenge for experts to develop new methods for rapid detection ofE.coli in the field of environmental monitoring and food safety.Electrochemical sensors and biosensors,an electrochemical analysis technology,develop very fast in the recent years.They have some advantages over otheranalytical systems in that they can operate in turbid media,offer comparableinstrumental sensitivity,and more amenable to miniaturization.Moreover,theapplication of nanomaterials in the preparation of electrochemical sensors hasprovided a new opportunity for their development.Combining with the biologicalcharacters of bacteria,the electrochemical sensors and biosensors have been widelyused to rapidly detect pathogens.In this dissertation,some sensitive and stable electrochemical sensors andbiosensors were fabricated based on novel nanomaterials,and were further applied torapidly detect E.coli in foods and water samples.Furthermore,an electrochemicalinstrument for the detection of bacteria was developed.Totally,this dissertation fallsinto five parts.Part 1:Preface (Chapter 1)In this part,we introduce briefly the biological characters and pathogenicity of E.coli in environment and attempt to review the current development of rapid detectionmethods of E.coli.Moreover,a critical regard to electrochemical sensors andbiosensors is given,and the application of electrochemical sensors to rapid detection of E.coli is also introduced.Part 2:Novel Nanomaterial Modified Electrode for Rapid detection of E.coli(Chapter 2 and 3)In the E.coli solution,β-D-galactosidase (β-gal) was released form E.coli by theeffect of permeabilizers,and catalyzed the hydrolysis ofp-Aminophenyl-β-D-galactopyranoside (PAPG) to produced p-aminophenol (PAP).So,the concentration of E.coli could be calculated by the current responses of PAPon the modified electrodes.In Chapter 2,a platinum nanoparticles chemical modified electrode (PtNP/GCE)was fabricated by electro-deposition and used to detect the concentration of E.coli.The influence of work potential,substrate concentration and permeabilizers wereinvestigated.Compared with conventional methods,the electrochemical technologycould be suitable for rapid detection of E.coli in the fields of food industry,environmental monitoring and clinic biomedicine.In Chapter 3,a multi-wall carbon nanotubes (MWNTs)/Nafion modified glassycarbon electrode (GCE) was fabricated for the rapid amperometric detection of E.coli.Due to the cation-exchange capacity of Nafion and the electrocatalytic ability ofMWNTs,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.colicould be detected after 4.5 h enrichment.The electrochemical sensor was further usedto detect E.coli in real sample,and the results were consistent to that obtained byplated count method.Part 3:The fabrication of tyrosinase (Tyr) biosensor and its application to rapiddetection of E.coli (Chapter 4 and 5)Biosensors have several advantages to other analytical methods in that they canoperate in turbid media,offer comparable instrumental sensitivity,and more amenableto miniaturization.In this part,two Tyr biosensors were fabricated based on novelnanomaterials and further applied to rapid detection ofE.coli.In Chapter 4,a Tyr biosensor was fabricated by immobilizing Tyr on the surface ofMWNTs-chitosan (Chit) composite modified glassy carbon electrode (GCE).TheMWNTs-Chit composite film provided a biocompatible platform for the Tyr to retain the bioactivity and the MWNTs possessed excellent inherent conductivity to enhancethe electron transfer rate.The Tyr/MWNTs-Chit/GCE biosensor showed highsensitivity (412 mA/M),broad linear response (1.0×10-8～2.8×10-5 M),low detectionlimit (5.0 nM) and good stability (remained 93% after 10 days) for determination ofphenol.Then,the biosensor was further applied to rapid detection of the E.coli.Thecurrent responses were proportional to the quantity of E,coli in the range of 104-107cfu/mL.After 5.0 h of incubation,E.coli could be detected as low as 10 cfu/mL.In Chapter 5,a novel tyrosinase (Tyr) biosensor was developed based on Fe3O4magnetic nanoparticles (MNPs)-coated carbon nanotubes (CNTs) nanocomposite andfurther applied to detect the concentration of coliforms with flow injection assay (FIA)system.Negatively charged MNPs were absorbed onto the surface of CNTs whichwere wrapped with cationic polyelectrolyte poly(dimethyldiallylammonium chloride)(PDDA).The Fe3O4 MNPs-coated CNTs nanocomposite was modified on the surfaceof the glassy carbon electrode (GCE),and Tyr was loaded on the modified electrodeby glutaraldehyde.The immobilization matrix provided a good microenvironment forretaining the bioactivity of Tyr,and CNTs incorporated into the nanocomposite led tothe improved electrochemical detection of phenol.The Tyr biosensor showed broadlinear response of 1.0×10-8～3.9×10-5 M,low detection limit of 5.0×10-9 M and highsensitivity of 516 mA/M for the determination of phenol.Moreover,the biosensorintegrated with a FIA system was used to monitor E.coli.The detection principle wasbased on determination of phenol which was produced by enzymatic reaction in the E.coli solution.Under the optimal conditions,the current responses obtained in FIAsystem were proportional to the concentration of bacteria ranging from 20 to 1×105cfu/mL with detection limit of 10 cfu/mL and the overall assay time of about 4 h.Thedeveloped biosensor with the FIA system was well suited for quick and automaticclinical diagnostics and water quality analysis.Part 4:Gold Nanolbels for Enhanced Electrochemical Immunoanalysis ofEscherichia coli (Chapter 6)In this part,a novel nanolabel based on gold nanoparticle modified with anti-E.coli peroxidase (HRP)-conjugated antibody is reported.It was used for enhancedelectrochemical immunoanalysis of E.coli.After the immuno-reaction,gold nanolabel,immunoparamagentic bead (IMB) and E.coli formed sandwich-typeimrnunocomplex.3,3,5,5-tetramethylbenzidine (TMB) was used as substrate for HRPin the presence of H2O2 and the enzyme activity of HRP was measured byelectrochemical detection coupled with flow injection assay (FIA).The concentrationof E.coli could be confirmed by amperometric response,which was in proportion tothe quantity of HRP on the sandwich-type immunocomplex.Gold nanoparticles wereused as carriers of antibodies resulting in the increase of antibodies attached to E.coli.So,the amperometric signal was enhanced and the detection sensitivity of E.coli wasimproved greatly.It showed a good linear relationship between amperometricresponses and the logarithmic value of E.coli concentration ranging from 1.0×102 to5.0×105 cfu/mL,with detection limit of 50 cfu/mL.The rapid detection could befinished within 1 h,shorter than other methods.Part 5:Development of electrochemical instrument for rapid detection ofbacteria (Chapter 7)In this part,we develop an electrochemical instrument for rapid detection ofbacteria by combining electrochemical techniques and biological characters ofbacteria.The instruments not only can detect total number of bacteria in the range of1.0×102 cfu/mL～1.0×105 cfu/mL,but also can detect the concentration of E.coliwithin 5 h in the range of 20～1.0×106 cfu/mL.The instrument was small,convenientand reliable.
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