Microfluidics for waterborne pathogen separation and detection

Abstract

There are millions of Cryptosporidium-attributable cases annually in children aged <24 months in the sub-Saharan Africa and India, Pakistan, Bangladesh, Nepal, Afghanistan regions, respectively, and ~202,000 Cryptosporidium-attributable deaths”. Improved monitoring is one solution to this challenge; however, detection of this pathogen is particularly challenging, particularly in regard to determining viability information. This thesis explores the development of novel protocols and devices for Cryptosporidium parvum (C. parvum), through usage of nanoparticles (NPs) and microfluidic methods. NP lysis approaches were developed as a low-cost, one-step rapid method with the possibility to then integrate lysis and molecular detection into one microfluidic device. Different materials, exposure times and concentrations were explored and ZnO NPs were found to be as effective as the traditional freeze-thaw protocol. Dielectrophoretic microfluidic devices were designed, prototyped and optimised for viability-based separations. Fabrication was attempted via laser ablation for the purpose of generating microchannels on PMMA sheets and Physical Vapour Deposition via an E-Beam system to investigate the deposition of electrodes; however, lift-off solvents were incompatible with PMMA. Electrode design modifications were implemented to optimise performance and efficacy of oocyst separation, based on viability, was assessed via an excystation assay where at the outlet collecting non-viable oocysts viability was found to be 5.9% and the other outlets showed a viability of 81.8% and 88.4%. The original sample provided had a viability of 89.7%. The work here adds to the growing number of studies investigating new ways of lysis and detection for C. parvum. The lysis and detection of the oocyst is currently highly intensive and a number of new methods of miniaturisation such as µPCR when integrated with a lysing and filtration device to create a more precise method of finding the presence of C. parvum in water samples. The DEP based separation device builds on the work of previous studies such as Su et al. and others in order to separate C. parvum based on its viability status in two different devices. This is a significant step showing that label-free separation of oocysts of the same species can be separated based on their viability.