A Point-of-Care Diagnostic Assay Utilizing the Hydrodynamics of an Evaporating Drop

Abstract

Point-of-care diagnostics suitable for the resource-limited environment should be low-cost and simple-to-use while maintaining a clinically meaningful performance capability. This thesis explores harnessing the hydrodynamics of an evaporating drop as one possible means of satisfying these design requirements. The microfluidic flow inside an evaporating drop has long been known to transport particles in solution to the edge of the drop and produce a characteristic ring pattern. In an initial proof-of-concept design using poly-L-histidine, a peptide mimic of the malaria biomarker pfHRPII, the presence of biomarkers caused self-assembly of a magnetic nanoparticle and a fluorescently-labeled micron-sized particle that were both functionalized with Ni(II)NTA. A small spherical magnet under the center of the drop prevented these assemblies from migrating to the drop’s edge while a non-reactive control particle flowed to the edge forming a ring pattern. This study showed that the presence or absence of biomarker resulted in distinctly different distributions of particles in the dried drop that can be visually detected as a shifted color pattern with a limit of detection of approximately 200-300nM. In a second study, optical coherence tomography was used to characterize three-dimensional flow patterns and evaluate the effects of substrate material, solution additives, and particle density on particle motion in evaporating drops. A revised assay design was tested in a third study that used these three-dimensional flow fields and an antibody-based biointerface to detect the presence of the bacteriophage M13K07 with a limit of detection of approximately 100fM. In this design, the bacteriophage triggered the self assembly of particles, which were transported to the center of the drop by Marangoni-induced eddy currents. The presence and size of a single spot distinguished a positive from a negative test. This research has shown that evaporating drops containing surface functionalized particles can be used as a simple means to detect the presence of target biomarkers. By demonstrating an antibody-based biointerface and sub-picomolar limit of detection, the assay design has the potential to be clinically useful for a wide range of disease targets.