Lithographically Fabricated Micro-Reflectors for Sensitive Point of Care Diagnostics

Timely diagnosis and treatment of infectious diseases calls for highly sensitive diagnostic assays. Traditional diagnostic methods include microscopy, polymerase chain reaction (PCR), immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA). Many of these diagnostic methods, however, require complex sample processing steps or labels (typically a dye, enzyme or fluor) which can suffer from low signal strength or photo-bleaching (of fluors), and can require elaborate instrumentation for their detection. Here, we propose several diagnostic platforms utilizing lithographically-fabricated, micron-scale, reflective particles as novel, optical labels conjugated to antibodies. The particles, in the shape of cubes, serve as ultra-bright labels which are easily detectable using inexpensive, low-numerical aperture optics. The cubes, unlike traditional labels, are not susceptible to photo-bleaching and do not require any signal amplification. In this research work, we investigate the use of these novel particulate labels for the development of new immunoassay formats which are simple-to-use, cheap and possess sensitivities comparable to or better than well-established clinical diagnostic methods. First, the fabrication and use of suspended, micro-fabricated retroreflector cubes as optical labels will be demonstrated. The cubes will be used in a novel flotation format in which a low density polymer module acts as a capture surface. Antibody-functionalized cubes, bridged to the capture surface by the target present in the sample, are floated to the top of the sample where they can be observed and counted. In a second assay format the cubes, which are engineered to be denser than the sample matrix, are allowed to mix with the sample and fall to a detection surface within a reaction vessel. Simply inverting the vessel allows non-specifically bound cubes to fall away leaving behind specifically bound particles. Lastly, the use of these reflective particles in a centrifugal diagnostics format will be presented. Centrifugal forces are tailored to improve signal to background ratios by removing non-specifically bound particles in a highly controllable way.