Development and Evaluation of Microwave-Accelerated and Metal-Enhanced Fluorescence Assays for Detection of Bacterial Pathogens
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Date
2016-01-01
Type of Work
Department
Chemistry & Biochemistry
Program
Chemistry
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This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
Distribution Rights granted to UMBC by the author.
Distribution Rights granted to UMBC by the author.
Abstract
Infectious diseases with high mortality rate or serious complications require rapid and accurate diagnosis. In order to develop rapid and sensitive assays for detection of bacterial pathogens, microwave-accelerated processes have been investigated to extract and fragment the bacterial DNA, followed by Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF)-based DNA detection. MAMEF combines the benefits of low-power microwave-acceleration (MA) with those of Metal-Enhanced Fluorescence (MEF) to aid in the development of ultra-fast and sensitive bioassays. The first part of this research explored the use of microwaves and highly-focused microwaves to rapidly extract and fragment DNA. Two different, but complementary approaches were investigated - the efficiency of microwaves for microbial lysing in comparison to conventional heating, and the effect of microwave-focusing metal structures on microbial lysing and DNA isolation/fragmentation. Using microwave irradiation, Neisseria gonorrhoeae was lysed and the DNA fragmented in as little as 30 seconds. Furthermore, the incorporation of microwave-focusing bowtie structures during the irradiation process enhanced the rate of cellular lysis and DNA fragmentation. The conditions used for the lysis of N. gonorrhoeae cannot be used to lyse other bacteria with different cell wall structure, such as the gram-positive Listeria monocytogenes. The second part of the research was devoted to the development and testing of MAMEF assays for the detection of the sexually transmitted infections chlamydia and gonorrhea and Salmonella infections. The chlamydia MAMEF assay which targets the cryptic plasmid proved to be more sensitive than the assay targeting the 16S rRNA gene. Additionally, both assays proved to have moderate sensitivity for detection of chlamydia directly from vaginal swabs. The gonorrhea MAMEF assay showed low sensitivity, and a new assay targeting a multi-copy gene has been developed. MAMEF-based detection of Salmonella from white blood cells-spiked samples and stool was also achieved. However, further work is necessary to develop a robust and reproducible assay. Lastly, proof-of-concept experiments were carried out to determine if a surface plasmon resonance (SPR) approach can be used to detect genetic modifications associated with antimicrobial resistance in gonorrhea. No reproducible results were obtained using a portable SPR instrument. In summary, the use of microwave-accelerated processes for detection of bacterial pathogens was demonstrated including the clinical validation of the chlamydia MAMEF assay.