| Escherichia coli O157:H7 is one of the most dangerous food born pathogens, and the illness due to its infection is often misdiagnosed and generally needs invasive and expensive medical tests before it is correctly diagnosed. The infectious dose of this bacterium is possibly fewer than 100 organisms, and primary sources for exposure to it include consumption of ground beef, sprouts lettuce, salami, unpasteurized milk, and juice contaminated by pathogens. Traditional methods for detection of this bacterium involve plating and culturing, enumeration methods, biochemical testing, microscopy, and flow cytometry, but these methods have not been satisfied with the needs of real application. Since the loss caused by E. coli O157:H7 is enormous in terms of medical cost and product recall, it is extremely urgent to develop some rapid and sensitive methods to detect this kind of bacteria in food or water supplies.Our research has developed some molecular biosensors for O157:H7 detection based on the immunoreaction and DNA hybridization sensing. Firstly, we adopted the quartz crystal microbalance (QCM) integrated with the self-assembled monolayers (SAMs) and advanced nanobiotechnologies to fabricate the QCM immunobiosensor and QCM DNA biosensor. Secondly, in order to explore the novel sensor and improve the sensing mechanism, we successfully developed the gold sensor array and an electrochemical DNA biosensor based on aluminum anodized oxide (AAO) nanopore membranes. The major contributions of this dissertation are summarized as follows:1. The sensitive and rapid detection of E. coli O157:H7 was realized by the QCM immunobiosensor processed on the home-made detection system. A polyelectrolyte layer-by-layer self-assembly (LBL-SA) method was proposed for rapid surface modification, which had shorten the fabrication time of the immunobiosensor.2. A gold nanoparticle-enhanced QCM DNA biosensor is operated by the flow-and-inject method. The inner Au nanoparticles were immobilized onto the thioled surface of the Au electrode, then more specific thiolated single-stranded DNA (ssDNA) probes could be fixed through Au-SH bonding. The outer avidin-coated Au nanoparticles could combine with the target DNA to increase the mass thus amplify the signal. The whole process has indicated that the combination of nanobiotechnologies and biosensing is a powerful means to improve the sensitivity of biosensor.3. A novel electrochemical immunobiosensor was developed for rapid multichannel detection of E. coli O157:H7 based on gold electrode array. The effect of self-assembled monolayers (SAMs) and the bacteria layers on the electrochemical properties of the electrodes were quantitatively analyzed in terms of constant phase element (CPE) and electron transfer resistance using proper equivalent circuit. The biosensor could detect six different concentrations of bacteria at the same time, which reduced the detection time to a large scale. With good sensitivity and high efficiency, this gold array could be integrated into some existing detection schemes for parallel detection.4. A nano-biosensor based on single-stranded DNA (ssDNA) probe functionalized aluminum anodized oxide (AAO) nanopore membranes was demonstrated for the bacteria DNA detection. An original and dynamic hybridization procedure was proposed in the existence of Taq DNA polymerase and dNTPs under controlled reaction temperature. The probe strands would be extended as long as the target DNA strands, and the capability to block the ionic flow in the pores has been prominently enhanced by the double-strand complex. This DNA biosensor could not only be applied for rapid label-free bacteria detection, but also be integrated into a self-contained biochip for various ss-DNA analysis.
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