The utilisation of point-of-contact diagnostic devices has evolved into a crucial cornerstone
within the global public health landscape. Notably, their significance became abundantly clear during
the COVID-19 pandemic. This doctoral thesis is dedicated to introducing and perfecting an innovative
point-of-contact system designed for the detection of specific genes. This system is rooted in a
modified recombinase polymerase DNA amplification method, complemented by an adaptable
nucleic acid lateral flow assay.
To showcase the capabilities of this novel system and elucidate the processes involved in its
design and optimization for a specific target gene locus, we employed genes linked to methicillinresistant Staphylococcus aureus as our detection targets. The system proved its effectiveness in
detecting the antibiotic resistance gene mecA, commonly found in methicillin-resistant
Staphylococcus aureus, even at a single genetic copy, all in less than an hour.
The research in this presentation provides a comprehensive breakdown of the procedures
necessary to effectively identify conserved gene loci suitable for our developed system. Additionally,
we delve into the methodology for designing compatible primer sets. To make the development of
this system possible, we established a kinetic model for real-time recombinase polymerase
amplification, which is presented herein. This model enabled us to explore the kinetics of recombinase
polymerase amplification reactions in real time, employing a custom-built solid-state fluorometer.
Modifications were made to the standard recombinase polymerase amplification procedures to
accommodate the altered primer system, with further adjustments detailed to ensure compatibility
with the developed nucleic acid lateral flow device. We also address the kinetic implications of these
modifications and outline a procedure for further optimising the recombinase polymerase
amplification reaction conditions to mitigate any performance losses.
The thesis goes on to discuss the sonochemical synthesis of monodisperse 21nm thiolfunctionalised silica-coated superparamagnetic nanoflowers. These nanoflowers were thoroughly
characterised using high-resolution transmission electron microscopy, X-ray diffraction, and UV-Vis
spectroscopy. Notably, they exhibited the remarkable ability to remain in colloidal solution for
extended periods and could endure freeze drying and reconstitution without requiring mechanical
redispersion. We also describe how these nanoflowers were to catalyse RPA reactions in solid phase,
in the presence of an alternating magnetic field. The magnetic field was generated using a battery-powered field generator developed in-house, compact enough to integrate into a standard lateral flow
device, akin to the ones currently available in the market, such as the ClearBlue Digital device.