Point-of-need biosensors for the detection of respiratory biomarkers

Abstract

Asthma is a chronic disease affecting over 300 million people worldwide. Airway inflammation is a central feature of asthma. Quantitative sputum cytometry is the most validated method to assess this and to adjust anti-inflammatory therapy, yet it is underutilized due to rigorous processing that requires expensive specialized technicians. To address these limitations, this thesis focuses on the development of several point of need biosensors that rapidly quantify respiratory biomarkers as alternatives to traditional laboratory tests. The project began by developing a paper based sensor for detection of myeloperoxidase (MPO), a neutrophil biomarker. A test was developed using commercially available antibodies, showing direct correlation between the test line colour intensity and total neutrophils. This work was expanded to include a second protein target, eosinophil peroxidase (EPX), for identification of eosinophils. Although the test performed well using neat samples, it failed to identify EPX in clinical sputum samples. Analyzing pre-treatment methods identified that a quick immunoprecipitation technique using protein A/G beads followed by syringe filtration allowed for the device to successfully quantify EPX, eliminating the need for a centrifuge. However, the limited supply of commercial anti-EPX antibodies combined with the need for sample pre-treatment prompted investigation into alternative detection avenues. Nucleic acid aptamers were explored, with aptamer selection for EPX producing several aptamer candidates. Binding affinity and specificity tests were performed, with the T1-5 aptamer emerging. T1-5 was capable of selectively binding EPX over MPO with high affinity. This aptamer was integrated into a simple pull-down assay, capable of detecting EPX with an order of magnitude lower limit of detection than the antibody test. Overall this work has developed multiple sensors with the potential to overcome the limitations of accessibility to sputum cytometry, rapidly identify the presence and type of airway inflammation, and deliver personalized treatment strategies that not only reduce the global healthcare burden, but also greatly improve a patient’s quality of life.