Design and development of a novel, self-contained, and field-portable biosensor platform for the detection and identification of microbial pathogens in near-real time.

Hall, James (2019) Design and development of a novel, self-contained, and field-portable biosensor platform for the detection and identification of microbial pathogens in near-real time. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The interactions of microorganisms are considered fundamental to all life, and plants and animals are increasingly viewed in the context of their associated microbiomes. However, there are significant health and economic costs associated with microbial pathogen contamination in farming, food processing, and the supply of potable water. While contemporary microbiology techniques play a valuable role in pathogen surveillance, they are predominantly laboratory-based, time-consuming, and are reliant on skilled personnel. To both ensure safety and productivity in increasingly intensive food production and to meet the challenge of antimicrobial resistance, there is a requirement for portable, real-time microbiology tools and a broader role for rapid diagnostics. This thesis outlines the development of a unique, self-contained, and fieldportable biosensor system for the detection of microbial DNA sequences in food, water, and environmental samples. The novel design utilises a ‘macro-fluidic’ model as basis for sample processing, reagent introduction, and analytical functionality and takes advantage of low-cost materials and electronics. Based on DNA hybridisation, the platform benefits from extensive resources accumulated in phylogenetic classification and the development of technologies such as gene sequencing and polymerase chain reaction (PCR). Other than sample loading, the biosensor system is totally automated and simple to use. An Arduino microprocessor controls all process control and electrochemical analysis, while user input and data display are handled by a smartphone app. The ground-up design makes use of modular design principles and off-the-shelf electronic components. The platform cell lysis unit demonstrates DNA extraction performance comparable to that of bench-top mini-prep kits, with high elution efficiency and repeatability. The macro-fluidic design is entirely novel and is unique in delivering high quality nucleic acid purification in a portable unit. The DNA amplification and hybridisation-based biosensor module demonstrates highly sensitive (pico-molar) nucleic acid detection and a lower limit of approximately 500 bacteria in the detection and identification of Campylobacter jejuni. The platform can generate samples for comparative analysis or for analysis using technologies such as the Minion portable gene sequencer. Using innovative design to bring self-contained molecular analysis into the field, the developed biosensor platform has potential applications in detection and identification of microbial species in areas from pathogen surveillance and microbial population characterisation to antibiotic resistance tracking and clinical diagnostics.