| Escherichia coli (E.coli) is a part of the normal microflora of the intestinal tract of humans and warm-blooded animals.It is widely distributed in the natural environment and most strains are nonpathogenic. However, some strains of E.coli can cause diarrhea, dysentery, gastroenteritis, food poisoning. Numerous examples of disease outbreaks due to contamination have been reported worldwide.Therefore, the number of coliform bacteria (or coliform values) is usually considered as hygiene standard in drinking water and food (or drugs). To aviod the negative effects caused by E.coli, the World Health Organization and health department in some countries develop appropriate regulations to limit the number of E.coli in the areas such as food, water and daily necessities.Conventional methods for the detection of E.coli are multiple-tube fermentation (MTF), membrane filter (MF), plate count and so on.For example, the membrane filter, which are laborious and time-consuming (at least 48 h), consuming many reagents.Thus, traditional methods are difficult to meet requirements on timely monitoring in the environment, food, health and other fields.Compared with traditional methods, developing more sensitive, rapid and specific methods to detect the E.coli become great challenges to scientists.The currently developed methods include enzymatic methods, molecular methods, immunological methods, etc. Moreover, modern instruments such as Matrix-assisted laser desorption/ionization mass spectrometry, GC-MS have been used for developing new methods to detect E.coli.Due to the unique catalytic properties and biological compatibility, nano-materials are widely used for designing ultra-high sensitivity, high selectivity of electrochemical sensors.Because of the high specificity and selectivity, immunoassay is widely used in clinical diagnosis, environmental analysis, drug analysis, food safety inspection and other fields.Electrochemical method has high sensitivity, simple operation, easy-to-device miniaturization and so on. Therefore, we combine nanotechnology, electrochemical immunoassay technology, to realize the detection of E.coli in water in high sensitivity. We synthesized the core-shell Cu@Au nanoparticles (NPs)as anti-E.coli antibody labels and applied it to detect E. coli. The method can detect E. coli with a detection limit of 30 cfu/mL. We also synthesized Fe3O4@Au Nanoparticles, and the Nanoparticles were used as Labels for Electrochemical Immunodetection of E.coli in Water. With preconcentration and pre-enrichment steps, it was possible to detect E.coli concentration as low as 2 cfu/mL in river water samples.An amperometric immunosensor for E.coli detection was developed by immobilizing anti-E.coli antibodies on poly-o-ABA/Au NPs modified GCE. The detection limit of this method to E.coli was 300 cfu/mL. Our work mainly focused on the following aspects:Part1.Development of an Electrochemical Immunoassay for Rapid Detection of E. coli Using Anodic Stripping Voltammetry Based on Cu@Au Nanoparticles as Antibody LabelsA sensitive electrochemical immunoassay for rapid detection of E. coli has been developed by Anodic Stripping Voltammetry (ASV) based on core-shell Cu@Au nanoparticles (NPs) as anti-E.coli antibody labels. The characteristics of Cu@Au NPs before and after binding with antibody were confirmed by transmission electron microscopy (TEM).After Cu@Au-labeled antibody reacted with the immobilized E. coli on PS-modified ITO chip, Cu@Au NPs were dissolved by oxidation to the metal ionic forms, and the released Cu2+ ions were determined at GC/Nafion/Hg modified electrode by ASV. The utilization of GC/Nafion/Hg modified electrode could enhance the sensitivity for Cu2+ detection with a concentration as low as 9.0×10-12 mol/L. Since Cu@Au NPs labels were only present when antibody reacted with E.coli, the amount of Cu2+ directly reflected the number of E.coli. The technique could detect E.coli with a detection limit of 30 CFU/mL and the overall analysis could be completed in 2 h. By introducing a pre-enrichment step, a concentration of 3 cfu/10 mL E.coli in surface water was detected by the electrochemical immunoassay. Part2. Fe3O4@Au Nanoparticles as Labels for Electrochemical Immunodet ection of E.coli in WaterFe3O4@Au nanoparticles were synthesized. The surface of nanoparticles was modified with horseradish peroxidase (HRP)-anti-E.coli antibody for amplified electrochemical immunoassay of E.coli. Poly-thionine (PTH)film was electro-polymerized on the GCE as an electron-transfer mediator. Gold nanoparticles (GNPs) on the PTH film constructed an effective antibody immobilization matrix. Using the typical sandwich type immmunoassay, E.coli could be detected according to the H2O2 reduction currents catalyzed by HRP labeled on anti-E.coli antibody. The method allowed quantitative determination of E.coli concentration from 50 to105 cfu/mL, with a detection limit of 20 cfu/mL. By introducing pre-enrichment steps, a concentration of 2 cfu/mL E.coli was detected with this method.Part3.Development of Amperometric Immunosensor for E.coli based on Gold nanoparticles and Poly-o-ABAAn alternative amperometric immunosensor was developed for the detection of E.coli.To construct the base of immunosensor, gold nanoparticles (Au NPs) were electrochemically deposited on the GCE and o-aminobenzoic acid was electropolymerized on the Au NPs with high porosity and hydrophilicity. The formed poly-o-ABA/Au NPs modified GCE was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared Spectrometry (FTIR).Anti-E.coli antibody was covalently immobilized on the poly-o-ABA/Au NPs modified GCE, followed by the E.coli in the sample, then recognized by the HRP-anti-E.coli antibody. The HRP at the immunosensor can catalyze amperometric immunoassay with H2O2 as substrate.Under optimal conditions, the amperometric currents were found to be proportional with logarithmic value of E.coli from 103 to 107 cfu/mL. The proposed immunosensor was successfully applied for real sample detection. |
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