Development of a novel electrochemical lab-on-a-disc, bio-sensors and bio-assays for pathogen capture and detection

The development of rapid pathogenic detection continues to be at the forefront of the greatest challenges of our clinical settings to date. Sepsis is a serious systematic inflammatory response which is triggered by bacterial, viral, or fungal infections. It persists to be a primary cause of patient death despite the medical advances made in therapeutics and antibiotics development. The non-specific symptoms of sepsis are the utmost limitation of diagnosis of the condition. Delays in detection therefore leads to an increase in patient mortality. Current gold standards within microbiology laboratories are too slow, therefore a huge demand exists for a reliable method for early stage detection of sepsis. In this work, the development of biosensors and point-of-care systems for use with electrochemical, centrifugal lab-on-a-disc, Enzyme Linked Oligonucleotide Assay (ELONA), Polymerase Chain Reaction (PCR) and Recombinase Polymerase Amplification (RPA) techniques are demonstrated. A novel integrated point-of-care LoaD system has allowed a real-life application of the sensors to be studied for hospital samples. This innovative system allows pre-loading of reagents and samples which are systematically triggered using spin-frequencies where EIS techniques are adopted to give a sample to answer in under 15 minutes. These devices are single use, therefore are ideal for quick, infectious sample testing. A full optimisation of the device was required as fluid manipulation of various samples is a key-feature where samples ranging from buffer to whole blood can be tested within the same device without the need for sample pre-treatment. A label-free electrochemical biosensor, capable of capture and detection of pathogens, was produced by using a self-assembled monolayer and antibody capture layer was fabricated and characterised to compare with the single use biosensor developed for this work. Real-cultured samples from a microbiological clinic were studied to determine the capabilities of the device for real point-of-care settings. The device, which is capable of detecting the presence of pathogens, and furthermore the category in which they lie in, has proved to be an exciting and critical step-forward in early stage sepsis detection. Further analysis allowed a specific threshold to be determined, where a change in 300 Ohms to the system signifies the presence of a pathogen on the capture surface due to changes in the interfacial capacitance of the biosensor. Using confocal microscopy, the surface coverage of the captured pathogens was determined, where a directly proportional relationship exists between the number of pathogens captured and the change in the impedance response. This highly novel system is unlike any other commercially available technology for pathogen detection. Other methods have been explored, such as ELONA and DNA amplification. With very low detection limits of 23 CFU/ml for the ELONA, 5 CFU/ml for the PCR assay and finally 9 CFU/ml for the RPA assay. With varying assay times of 195-103 minutes, the assays were highly capable of detecting extremely low counts of E. coli in clean buffer samples. Although laborious they have also been proved to be able to detect specific E. coli pathogens which may lead the way for the development of a secondary analysis tool to specifically identify the causative pathogen for a highly precise diagnosis for patients