Electrophoretic immunoassays for oral diagnostics

Analysis of oral fluid (e.g., saliva) has the potential to aid diagnosis of both oral and systemic diseases. Oral fluids offer advantages over other bodily fluids, including quick, non-invasive, and inexpensive collection in both clinical and non-clinical settings. Nevertheless, current assay techniques using saliva are time-consuming, require large sample volumes, and are not amenable to automation or portability. We report on a robust microfluidic platform that performs rapid immunoassay-based analysis (<3 minutes) of saliva with low sample volume requirements (10 μL) and appreciable sensitivity (10^-12 M) with a high-degree of autonomy. Immunoassays for the detection of the systemic disease biomarker, C-reactive protein, and the cytokines interleukin-6 and tumor necrosis factor-α were conducted in spiked human saliva using a microfluidic chip. The immunoassay developed is based on binding of fluorescently-labeled reporter antibodies to analytes followed by electrophoretic separation and quantitation of bound and unbound reporter. The separation selectivity was achieved using native polyacrylamide gel electrophoresis in a microchip containing in situ photolithographically patterned cross-linked polyacrylamide. Native electrophoresis conditions maintained the integrity of fragile immune complexes and provided rapid separations of complex samples. On-going work focuses on integration of the on-chip immunoassay with miniaturized hardware towards developing a portable prototype.