Application And Research Of Nanomaterials-Based Electrochemical DNA Biosensor In The Rapid Detection Of Escherichia Coli

Escherichia coli (E. coli), found in large numbers in the intestines of human and other warm-blooded animals, spread abroad in natural environment and contaminate water supplies and recreational waters. Most of E. coli may not cause diseases, while some of them produce enterotoxin and are the major cause of infection outbreaks with serious consequences. In particular, the serotype O157:H7 associates with several human diseases including diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome and even septicemia. 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 microbiological detection of coliforms includes plate counting, multiple-tube fermentation and membrane filter. These methods are accurate, but they were not suitable for rapid detection of contamination because of their long detection period and complex operation. More recently, a number of new methods have been developed based on different measuring principles, such as polymerase chain reaction (PCR), immunoassay, electrochemical method and fluorescence assay. 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. Therefore, the development of rapid, sensitive, simple and inexpensive detection methods of E. coli remains a challenge to many scientists and engineers.Recently, more and more nanostructure materials were applied in analytical chemistry. A sensitive electrochemical DNA biosensor based on a mixed monolayer structure self-assembled at nanoporous gold (NPG) electrode surface was prepared for E. coli detection, with detection limit of 50 cfu/mL. Due to the good biocompatibility of the magnetic nanoparticles (MNPs), a rapid, specific and sensitive method for assay of E. coli using Fe2O3@Au core/shell nanoparticles in combination with DNA hybridization technique was proposed, with low detection limit of 5 cfu/mL. An electrochemical DNA biosensor based on nanogold/PDDA-MWNTs was developed to detect the concentration of E. coli and the detection limit was 5 cfu/mL. The dissertation includes four sections. Chapter one:IntroductionBasic knowledge on E. coli was introduced including biological feature, pathogenicity and sanitation standard. Conventional methods and some novel methods for detection of E. coli were demonstrated. What' more, excellent properties, preparation and characterization of nanostructure materials as well as the application of nanostructure materials on rapid detection of E. coli were presented.Chapter two:A Sensitive Nanoporous Gold-based Electrochemical DNA Biosensor for Escherichia coli DetectionA sensitive electrochemical DNA biosensor based on a mixed monolayer structure self-assembled at nanoporous gold (NPG) electrode surface was prepared for Escherichia coli (E. coli) detection. NPG was fabricated on gold electrode, onto which thiolated oligonucleotides (SH-DNA) and mercaptohexanol (MCH) were covalently linked forming a mixed self-assembled monolayer (SAM). The hybridization between the SH-DNA/MCH modified biosensor and E. coli DNA was monitored with differential pulse voltammetry measurement using methylene blue (MB) as the hybridization indicator. The biosensor can detect 1×10-12 M DNA target and 50 cfu/μLE. coli without any nucleic acid amplification steps. The detection limit was lowered to 50 cfu/mL after 5.0 h of incubation.Chapter three:Fe2O3@Au Core/Shell Nanoparticle-Based Electrochemical DNA Biosensor for Escherichia coli DetectionA Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe2O3@Au nanoparticles were prepared by reducing HAuCl4 on the surfaces of Fe2O3 nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.Chapter four:Nanogold/PDDA-MWNTs/GCE Electrochemical DNA Biosensor for Rapid Detection of Escherichia coliThe nanogold/PDDA-MWNTs assemblies were prepared using an electrostatic layer-by-layer (LBL) technique. This DNA biosensor is based on a sandwich detection strategy involving capture probes immobilized on the assemblies, target and reporter probes labeled with glucose oxidase. The sequence-specific DNA sensing was accomplished by the electrochemical detection of H2O2 (byproduct of enzyme reaction). The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 1000 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be further lowered to 10 cfu/mL after 4.0 h of incubation.