Novel microfluidic approaches for rapid point-of-care quantitation of Escherichia coli using immunoassays

There is a major clinical demand for rapid diagnostic tests capable of detecting pathogens in human samples. This is driven by an unprecedented clinical need in prescribing antibiotics more objectively and fighting antimicrobial resistance. Despite the remarkable breakthroughs, miniaturisation of such bioassays is challenging and expensive.

Urinary Tract Infections are the most common bacterial infection, with Escherichia coli (E. coli) being responsible for >80% of cases, presenting concerning resistance levels to the last generation of antibiotics. Currently, diagnostics industry struggle to deliver miniaturised biosensors capable of detection and identifying bacteria at format compatible with point-of-care.

This thesis presents novel experimental insights for sensitive and rapid detection of E. coli using polyclonal antibodies immobilized on the inner surface of an inexpensive 10-bore FEP-Teflon® MicroCapillary Film. Overall the results achieved allowed identification of three major challenges for miniaturisation of bacteria detection: the interrogation volume, gravity and shear stress. Coated capillaries acted as a very high affinity system, capturing up to 100% of E. coli cells, with clear evidence of immunospecificity. In addition, an opportunity to explore the ‘open’ fluidics aspect of microcapillary systems to concentrate bacteria from a large sample volume was identified and therefore speed up the detection of bacteria in clinical samples without the need for microbiological incubation. Therefore, this enabled the development of a fluorescence sandwich immunoassay capable of quantifying E. coli in buffer and synthetic urine in less than 25 minutes, yielding a limit of detection (LoD) up to 240 CFU/ml, commensurate with the cut-off value required for UTIs (10³-10⁵ CFUs/ml). This proof-of-concept was achieved exploiting low cost readout systems such as a smartphone and demonstrated reproducible and robust performance in synthetic urine. Ultimately, two new gravity-driven microfluidic devices, denominated MCF-funnel and MCF-siphon, were designed and manufactured to address autonomy, power-free and portability requirements.