Abstract
Immunoassays are utilized for a wide variety of clinical and biomedical research applications. In typical immunoassays, analytes are captured, labeled, and quantified on a single surface (e.g., the bottom of a well plate). In order to minimize the background, this type of assay must be washed multiple times between each of these steps to ensure residual reagents (e.g., unbound labeling antibody) are removed from the system. In this manuscript, the immunoassay is fundamentally reconfigured, such that each reagent is confined to its own well and no wash steps are required. Using immiscible filtration assisted by surface tension (IFAST), a technique developed for nucleic acid and whole cell purifications, immunoassays can be drastically simplified such that all reagent manipulation is performed at the start of the assay (i.e., no pipetting steps are necessary during the assay). Analytes are bound to paramagnetic particles via antibodies and drawn through oil barriers between four isolated compartments: (1) sample well, (2) primary antibody labeling well, (3) secondary antibody labeling well, and (4) readout buffer well. Using this technique, we have demonstrated repeatable detection of as little as 188 fg of protein. IFAST immunoassay functionality is demonstrated by detecting a well accepted prostate cancer biomarker, prostate specific antigen (PSA). Assay performance was assessed by measuring known concentrations of recombinant PSA protein. The assay was then used to measure PSA concentrations in conditioned media and human plasma samples.