Approximately 3.4 million deaths occur each year due to lack of clean water. The Shigatoxigenic group of Escherichia coli (STEC) bacteria causes approximately 2.2 million of these deaths and nearly a billion cases of illness. Current approved testing methods for Escherichia coli require a 12-24 hours growth of the sample in order to test for an indicator species. This indicator species only suggests possible E. coli O157:H7 presence, but does not confirm it. Direct culture testing cannot identify the pathogenic STEC bacteria either. Confirmation of toxicity requires molecular methods of identification leading to specialized equipment needs and higher testing costs. Typical molecular methods use polymerase chain reaction (PCR) to amplify a very specific target DNA sequence, indicating the presence of a specific microorganism. PCR requires a thermocycler and a clean laboratory environment. Recent work has been successfully explored using nanomaterial based systems to identify the same genes used in a PCR reaction in a simpler detection system. The nanomaterials are used for both signal amplification and as the detection molecule. In this dissertation work a biosensor utilizing nanoparticles has been developed for the detection of Shiga-like toxin 1 gene that is present in Escherichia coli O157:H7. The system has been successfully constructed using a novel carbohydrate coated gold nanoparticle for delivery of the self assembling co-polymerization DNA oligonucleotide reporters. A set of self-assembled single-stranded DNA (ssDNA) molecules has been developed to amplify a target sequence without the use of enzymes. The full detection system incorporates the gold nanoparticle and a magnetic microparticle into a DNA recognition step. The gold nanoparticle is used for both co-polymerization detection and electrochemical detection. Input material (genomic DNA) is extracted using a modified commercial extraction method to produce PCR quality DNA from whole bacterial cells. When extraction and recognition elements are combined, a limit of detection for E. coli O157:H7 of 105 cells/mL using co-polymerization and 101 cells/mL with electrochemical detection has been achieved. Sample preparation from spiked culture samples until final detection takes a total of 7 hours. Electrochemical detection provides only a presence or absence indicator, where tethered co-polymerization is able to provide quantitative values of the input bacterial concentration. So a combination of co-polymerization amplification and electrochemical detection can provide the potential for more sensitive and quantitative measurement without the need for enzymes as in PCR applications.
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