Rapid Pathogen Detection using Handheld Optical Immunoassay and Wire-guided Droplet PCR Systems
AuthorYou, David Jinsoo
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PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThis work introduces technology for rapid pathogen detection using handheld optical immunoassay and wire-guided droplet PCR systems. There have been a number of cases of foodborne or waterborne illness among humans that are caused by pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, Influenza A H1N1, and the norovirus. The current practices to detect such pathogenic agents are: cell/viral culturing, immunoassays, or polymerase chain reactions (PCRs). These methods are essentially laboratory-based methods that are not at all real-time and thus unavailable for early-monitoring of such pathogens. They are also very difficult to be implemented in field, preventing early detection opportunities. This dissertation is divided into three papers that present methodologies towards the expeditious detections of infectious pathogens and the miniaturization of these detection systems towards field-deployable and point-of-care applications. Specifically, the work presented focuses on two methodologies: (1) light scatter detection using immunoagglutination assays with optimized Mie light scatter parameters in a real biological matrix consisting of plant tissue, and (2) wire-guided droplet manipulations for rapid and improved sample analysis, preparation, and PCR thermocycling. Both of these methods carry a collective objective towards providing high impact technologies for addressing the issues of food-related outbreaks and overall public safety. In the first paper, the direct and sensitive detection of foodborne pathogens from fresh produce samples was accomplished using a handheld lab-on-a-chip device, requiring little to no sample processing and enrichment steps for a near-real-time detection and truly field-deployable device. The detection of Escherichia coli K12 and O157:H7 in iceberg lettuce was achieved utilizing optimized Mie light scatter parameters with a latex particle immunoagglutination assay. The system exhibited good sensitivity, with a limit of detection of 10 CFU mL⁻¹ and an assay time of <6 min. Minimal pretreatment with no detrimental effects on assay sensitivity and reproducibility was accomplished with a simple and cost-effective KimWipes filter and disposable syringe. Mie simulations were used to determine the optimal parameters (particle size d, wavelength λ, and scatter angle θ) for the assay that maximize light scatter intensity of agglutinated latex microparticles and minimize light scatter intensity of the tissue fragments of iceberg lettuce, which were experimentally validated. This introduces a powerful method for detecting foodborne pathogens in fresh produce and other potential sample matrices. The integration of a multi-channel microfluidic chip allowed for differential detection of the agglutinated particles in the presence of the antigen, revealing a true field-deployable detection system with decreased assay time and improved robustness over comparable benchtop systems. In the second paper, we demonstrate a novel method of wire-guided droplet manipulations towards very quick RT-PCR. Because typical RT-PCR assays take about 1–2 h for thermocycling, there is a growing need to further speed up the thermocycling to less than 30 min. Additionally, the PCR assay system should be made portable as a point- of-care detection tool. Rapid movements of droplets (immersed in oil) over three different temperature zones make very quick PCR possible, as heating/cooling will be made by convective heat transfer, whose heat transfer coefficients are much higher than that of conduction, the latter of which is used in most conventional PCR systems. A 30-cycle PCR of a 160 bp gene sequence amplified from 2009 H1N1 influenza A (human origin) was successfully demonstrates in 6 min and 50 sec for a very large 10 μL droplet (with additional 4 min for reverse transcription). The proposed system has a potential to become a rapid, portable, point-of-care tool for detecting influenza A. In the third paper, a wire-guided CNC apparatus was used to perform droplet centrifugation, DNA extraction, and VQ-PCR thermocycling on a single superhydrophobic surface measuring 25 mm by 55 mm and a multi-chambered PCB heater. This methodology exhibited no limitations on the complexity and configuration of procedures that it can perform, making it versatile and far-reaching in its applications. The only modification required for adding or implementing changes for a new protocol is through simple pre-defined programming. The highly adaptive and flexible system was used to execute easily pre-programmed droplet movements and manipulations for the rapid detection of Escherichia coli from PCR detection. Serial dilutions were performed to simulate a diluted field sample with a high level of accuracy. Centrifugation of the diluted sample containing E. coli demonstrated a novel approach to sample pre-treatment. Furthermore, the extraction of DNA from the sample droplet containing E. coli was also performed on the same superhydrophobic surface as the previous 2 steps, requiring less than 10 min. Following extraction, the genetic material was amplified using wire-guided droplet PCR thermocycling, successfully completing 30 cycles of Peptidase D (a long 1500 bp sequence) in 10 min. The droplet centrifugation process was determined to greatly improve the positive band intensity over the non-centrifuged sample. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.
Degree ProgramGraduate College
Agricultural & Biosystems Engineering