The development of a novel point-of-care instrument and microfluidic immunoassays for the multiplexed measurement of cardiac biomarkers

The early diagnosis of cardiovascular diseases is crucial in minimising the extent of injury sustained during acute cardiac events. Numerous cardiac biomarkers reflect damage of the myocardium, however the cardiac troponins are recognised as the gold standard biomarkers of acute coronary syndrome, with their measurement using highly sensitive immunoassays fundamental to the implementation of accelerated diagnostic pathways. Combining the measurement of certain cardiac biomarkers with established risk stratification algorithms can enhance profiling of individuals at greater risk of experiencing acute cardiac injuries. Furthermore, risk profiling can be improved by incorporating multiple biomarkers in a multiplexed detection scheme. This thesis details the development of a novel point-of-care (POC) immunoassay automation instrument and accompanying microfluidic immunoassays for the multiplexed measurement of cardiac troponin I (cTnI), heart-type fatty acid binding protein (H-FABP) and myeloperoxidase (MPO). The standalone instrument utilises a low-cost micro camera to transmit ‘real-time’ video footage to a smart phone device for liquid flow visualisation and colourimetric measurements of ‘on-disc’ liquids. System vibration analysis was conducted to establish the safe operating speed range of the POC instrument, whilst colourimetric measurement accuracy was validated against a laboratory instrument. A centrifugal microfluidic disc was designed to host the immunoassays, with the function of key fluidic structures—including a bespoke Coriolis switch—investigated and optimised to improve control over ‘on-disc’ liquids and to minimise individual fluidic structure malfunction. As direct immobilisation of bioreceptors on internal reaction reservoir surfaces was implemented prior to performing the microfluidic immunoassays, novel dual�material reservoir sealing devices were designed and fabricated using a stereolithography 3D printer. This unique localised immobilisation approach ensured assay reagents were conserved and channel surface wetting properties remained unaltered following bioreceptor immobilisation. The microfluidic immunoassays were conducted using the novel POC instrument, however ultimately, time constraints resulted in the successful measurement of cTnI only. Moreover, very high concentrations (250 and 1,000 ng/mL) of cTnI were measured, with the colourimetric response variability considerably increased in comparison to the benchtop immunoassays. The siphon valve was chiefly responsible for the malfunction of the microfluidic immunoassays and contributed to the inability to measure H-FABP and MPO. Nonetheless, the parallel measurement of cTnI within 22 minutes using the novel POC immunoassay instrument demonstrated the principle capabilities of the system